Methods of treating rapidly progressive glomerulonephritis using chimeric and humanized anti-histone H4 antibodies

ABSTRACT

The present invention concerns chimeric or humanized antibodies or antigen-binding fragments thereof that comprise specific CDR sequences, disclosed herein. Preferably, the antibodies or fragments comprise specific heavy and light chain variable region sequences disclosed herein. More preferably, the antibodies or fragments also comprise specific constant region sequences, such as those associated with the nG1m1,2 or Km3 allotypes. The antibodies or fragments may bind to a human histone protein, such as H2B, H3 or H4. The antibodies or fragments are of use to treat a variety of diseases that may be associated with histones, such as autoimmune disease (e.g., SLE), atherosclerosis, arthritis, rheumatoid arthritis, edema, sepsis, septic shock, hyperinflammatory disorder, infectious disease, inflammatory disease, immune dysregulatory disorder, GVHD, transplant rejection, atherosclerosis, asthma, a coagulopathy, myocardial ischemia, thrombosis, nephritis, inflammatory liver injury, acute pancreatitis, ischemia-reperfusion injury, stroke, cardiovascular disease, and burn.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/180,646, filed Feb. 14, 2014, which claims the benefit under 35U.S.C. 119(e) of provisional U.S. Patent Application Ser. No.61/765,150, filed Feb. 15, 2013.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 10, 2014, isnamed IMM342US1 SL.txt and is 71,342 bytes in size.

FIELD OF THE INVENTION

The invention relates to compositions and methods of use of anti-histoneantibodies or antigen-binding fragments thereof. In particularembodiments, the antibodies bind to human histones H2B, H3 or H4. Theanti-histone antibodies are of use for diagnosis and/or therapy of awide range of disease states, including but not limited to, autoimmunedisease (such as systemic lupus erythematosus), atherosclerosis,arthritis, rheumatoid arthritis, juvenile arthritis; edema; sepsis;septic shock; inflammation; non-septic hyperinflammatory disorder;infectious disease; thrombosis; nephritis; inflammatory liver injury;traumatic hemorrhage; acute pancreatits; acute respiratory distresssyndrome; ischemic injury; ischemia-reperfusion injury; ischemic stroke;cardiovascular disease; atherosclerosis; radiotherapy toxicity; cytokinetherapy toxicity; granulomatous disease; asthma; graft-vs.-host disease,cachexia, a coagulopathy; cancer; or burn effects and complicationsthereof. More particular embodiments may concern chimeric or morepreferably humanized forms of the anti-histone antibodies.

BACKGROUND

Sepsis is a major medical and economic burden to our society, affectingabout 700,000 people annually in the United States, causing over 200,000deaths annually, and costing approximately $16.7 billion per year (Anguset al., Crit Care Med 2001; 29:1303-1310; Martin et al., N Engl J Med2003; 348:1546-1554). The definition of sepsis has been difficult, andhistorically it was defined as the systemic host response to aninfection. A discussion of the clinical definition of sepsis,encompassing systemic inflammatory response syndrome (SIRS), sepsis perse, severe sepsis, septic shock, and multiple organ dysfunction syndrome(MODS) is contained in Riedmann et al., J Clin Invest 2003; 112:460-467.Since it has been a common belief that sepsis is caused by the host'soverwhelming reaction to the invading microorganisms, and that thepatient is more endangered by this response that than the invadingmicroorganisms, suppression of the immune and inflammatory responses wasan early goal of therapy.

Numerous and diverse methods of immunosuppression or of neutralizingproinflammatory cytokines have proven to be unsuccessful clinically inpatients with sepsis and septic shock anti-inflammatory strategies. (JClin Invest 2003; 112:460-467; Van Amersfoort et al. (Clin Microbiol Rev2003; 16:379-414), such as general immunosuppression, use ofnonsteroidal anti-inflammatory drugs, TNF-α antibody (infliximab) or aTNF-R:Fc fusion protein (etanercept), IL-1 (interleukin-1) receptorantagonist, or high doses of corticosteroids. However, a success in thetreatment of sepsis in adults was the PROWESS study (Human ActivatedProtein C Worldwide Evaluation in Severe Sepsis (Bernard et al., N EnglJ Med 2001; 344:699-709)), showing a lower mortality (24.7%) than in theplacebo group (30.8%). This activated protein C (APC) agent probablyinhibits both thrombosis and inflammation, whereas fibrinolysis isfostered. Friggeri et al. (2012, Mol Med 18:825-33) reported that APCdegrades histones H3 and H4, which block uptake and clearance ofapoptotic cells by macrophages and thereby contribute to organ systemdysfunction and mortality in acute inflammatory states. Van Amersfoortet al. state, in their review (ibid.) that: “Although the blocking ormodulation of a number of other targets including complement andcoagulation factors, neutrophil adherence, and NO release, are promisingin animals, it remains to be determined whether these therapeuticapproaches will be effective in humans.” This is further emphasized in areview by Abraham, “Why immunomodulatory therapies have not worked insepsis” (Intensive Care Med 1999; 25:556-566). In general, although manyrodent models of inflammation and sepsis have shown encouraging resultswith diverse agents over the past decade or more, most agents translatedto the clinic failed to reproduce in humans what was observed in theseanimal models, so that there remains a need to provide new agents thatcan control the complex presentations and multiple-organ involvement ofvarious diseases involving sepsis, coagulopathy, and certainneurodegenerative conditions having inflammatory or immune dysregulatorycomponents.

More recent work on immunoglobulins in sepsis or septic shock has beenreported. For example, Toussaint and Gerlach (2012, Curr Infect Dis Rep14:522-29) summarized the use of ivIG as an adjunct therapy in sepsis.The metanalysis failed to show any strong correlation between generalimmunoglobulin therapy and outcome. LaRosa and Opal (2012, Curr InfectDis Rep 14:474-83) reported on new therapeutic agents of potential usein sepsis. Among other agents, anti-TNF antibodies are in currentclinical trials for sepsis, while complement antagonists have shownpromising results in preclinical models of sepsis. Nalesso et al. (2012,Curr Infect Dis Rep 14:462-73) suggested that combination therapies withmultiple agents may prove more effective for sepsis treatment. Theimmunopathogenesis of sepsis has been summarized by Cohen (2002, Nature420:885-91).

The immune system in sepsis is believed to have an early intenseproinflammatory response after infection or trauma, leading to organdamage, but it is also believed that the innate immune system oftenfails to effectively kill invading microorganisms (Riedmann and Ward,Expert Opin Biol Ther 2003; 3:339-350). There have been some studies ofmacrophage migration inhibitory factor (MIF) in connection with sepsisthat have shown some promise. For example, blockage of MIF or targeteddisruption of the MIF gene significantly improved survival in a model ofseptic shock in mice (Calandra et al., Nature Med 2000; 6:164-170), andseveral lines of evidence have pointed to MIF as a potential target fortherapeutic intervention in septic patients (Riedmann et al., citedabove). Bucala et al. (U.S. Pat. No. 6,645,493 B1) have claimed that ananti-MIF antibody can be effective therapeutically for treating acondition or disease caused by cytokine-mediated toxicity, includingdifferent forms of sepsis, inflammatory diseases, acute respiratorydisease syndrome, granulomatous diseases, chronic infections, transplantrejection, cachexia, asthma, viral infections, parasitic infections,malaria, and bacterial infections, which is incorporated herein in itsentirety, including references. The use of anti-LPS (lipopolysaccharide)antibodies alone similarly has had mixed results in the treatment ofpatients with septic shock (Astiz and Rackow, Lancet 1998;351:1501-1505; Van Amersfoort et al., Clin Microbiol Rev 2003;16:379-414.

While both LPS and MIF have been pursued as targets in the treatment ofsepsis and septic shock, approaches which target LPS or MIF alone by anantibody have not been sufficient to control the diverse manifestationsof sepsis, especially in advanced and severe forms. Similarly, use ofcytokines, such as IL-1, IL-6 (interleukin-6), IL-8 (interleukin-8),etc., as targets for antibodies for the treatment of sepsis and othercytokine-mediated toxic reactions, has not proven to be sufficient for ameaningful control of this disease. Therefore, in addition to the needto discover additional targets of the cytokine cascade involved in theendogenous response in sepsis, it has now been discovered that bi- andmulti-functional antibodies targeting at least one cytokine or causativeagent, such as MIF or lipopolysaccharide (LPS), is advantageous,especially when combined with the binding to a host cell (or itsreceptor) engaged in the inflammatory or immune response, such as Tcells, macrophages or dendritic cells. Antibodies against an MHC classII invariant chain target, such as CD74, have been proposed by Bucala etal. (US 2003/0013122 A1), for treating MIF-regulated diseases, andHansen et al. (US 2004/0115193 A1) proposed at least one CD74 antibodyfor treating an immune dysregulation disease, an autoimmune disease,organ graft rejection, and graft-versus-host disease. Hansen et al.describe the use of fusion proteins of anti-CD74 with other antibodiesreacting with antigens/receptors on host cells such as lymphocytes andmacrophages for the treatment of such diseases. However, combinationswith targets other than CD74 are not suggested, and the disclosurefocuses on a different method of immunotherapy. Similar targets are alsouseful to treat atherosclerotic plaques (Burger-Kentischer et al.,Circulation 2002; 105:1561-1566).

In the treatment of infectious, autoimmune, organ transplantation,inflammatory, and graft-versus-host (and other immunoregulatory)diseases, diverse and relatively non-specific cytotoxic agents are usedto either kill or eliminate the noxient or microorganism, or to depressthe host's immune response to a foreign graft or immunogen, or thehost's production of antibodies against “self,” etc. However, theseusually affect the lymphoid and other parts of the hematopoietic system,giving rise to toxic effects to the bone marrow (hematopoietic) andother normal host cells. Particularly in sepsis, where animmunosuppressed status is encountered, use of immunosuppressivetherapies would be counter-indicated, so it is a goal to effect acareful balance between targeting and inhibiting key cells of theadaptive immune system while not depleting those involved with the hostmaintaining an active immune system.

A need exists for improved, more selective therapy of cancer and diverseimmune diseases, including sepsis and septic shock, inflammation,atherosclerosis, cachexia, graft-versus-host, and other immunedysregulatory disorders.

SUMMARY

Certain embodiments concern chimeric or humanized versions of antibodiesagainst histones, such as IMMU-H4, IMMU-H3 or IMMU-H2B. The amino acidsequences of the variable region domains of the IMMU-H4, IMMU-H3 andIMMU-H2B antibodies were determined by DNA sequencing. Chimericantibodies were designed and constructed by replacing murine constantregion sequences with human antibody constant region sequences.Humanized antibodies are designed and constructed by inserting theidentified CDR sequences into human antibody framework region (FR)sequences, attached to human antibody constant region sequences. Inpreferred embodiments, selected human FR amino acid residues arereplaced with the corresponding murine FR residues from the parentalmurine antibody, to optimize binding or other activities of thehumanized antibody.

The chimeric and/or humanized anti-histone antibodies or antigen-bindingfragments thereof are of use for diagnosis and/or therapy of a widerange of disease states, including but not limited to, autoimmunedisease, such as systemic lupus erythematosus (SLE), autoimmune diseaseother than SLE, atherosclerosis, arthritis, rheumatoid arthritis,juvenile arthritis; edema; sepsis; septic shock; inflammation; anon-septic hyperinflammatory disorder; infectious disease; thrombosis;nephritis; inflammatory liver injury; traumatic hemorrhage; acutepancreatits; acute respiratory distress syndrome; ischemic injury;ischemia-reperfusion injury; ischemic stroke; cardiovascular disease;atherosclerosis; radiotherapy toxicity; cytokine therapy toxicity;granulomatous disease; asthma; graft-vs.-host disease, cachexia, acoagulopathy; cancer; or burn effects and complications.

In certain preferred embodiments, a combination of anti-histoneantibodies may be used. Antibodies against human histones H1, H2A, H2B,H3 or H4 may be used in any combination. Other non-antibody therapeuticagents targeted against either histones or downstream effectors of ahistone-mediated pathway may also be utilized in combination withanti-histone antibodies or fragments thereof, administered eitherbefore, simultaneously with, or following administration of one or moreanti-histone antibodies or fragments thereof. Various therapeutic agentsof use in treating histone-associated disease states are known in theart, such as activated protein C (APC), thrombomodulin, a peptidefragment of histone H1, H2A, H2B, H3 or H4, granzyme A, granzyme B,plasmin, Factor 7-activating protease, heparin, and any such known agentmay be utilized in combination with the subject anti-histone antibodiesor antibody fragments. A human histone H4 peptide may comprise residues50-67 or 40-78 of human H4 (see, e.g., U.S. Publ. No. 20090117099).

In alternative embodiments, the disclosed methods and/or compositionsmay utilize one or more chimeric, humanized or human antibodies orantigen-binding antibody fragments that compete for binding with, orbind to the same epitope as, an IMMU-H4, IMMU-H3 or IMMU-H2B antibody.These can be combined with agents affecting or inhibiting the innate oradaptive immune systems (including proinflammatory effector cytokines ora proinflammatory effector chemokines; regulatory T cells and otherhematopoietic cells implicated in the disease); the complement system,and/or a coagulation factor or factors that contribute to the pathologyor pathogenesis of the disease. These combinations or multispecificagents, including multispecific antibodies, are intended to enhance theeffects of anti-histone antibodies in the management of these diversediseases.

Specific embodiments concern chimeric and humanized antibodies ofparticular allotypes. Preferably, the antibody constant region sequencesare selected to correspond to an nG1m1,2 heavy chain null allotype, morepreferably a G1m3 heavy chain allotype, more preferably a Km3 lightchain allotype.

Surprisingly, it is discovered that the chimeric and humanized forms ofthe anti-histone antibodies may exhibit a higher affinity for the targethistones than the parent murine antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate preferred embodimentsof the invention. However, the claimed subject matter is in no waylimited by the illustrative embodiments disclosed in the drawings.

FIG. 1. Comparison of the variable region amino acid sequences of themurine IMMU-H4 heavy and light chains with the corresponding sequencespublished by Monestier et al. (1993, Mol. Immunol 30:1069-75) for theBWA-3 antibody. Underlined residues show discrepancies. “X” indicates aresidue missing from the published sequence. The correct amino acidsequences of murine IMMU-H4 heavy chain (SEQ ID NO:98) and light chain(SEQ ID NO:99) are shown alongside the incorrect published sequences ofmurine BWA-3 heavy chain (SEQ ID NO:120) and light chain (SEQ ID NO:121)

FIG. 2. Comparison of the variable region amino acid sequences of themurine IMMU-H3 heavy and light chains with the corresponding sequencespublished in Monestier et al. (1993, Mol. Immunol 30:1069-75) for theLG2-1 antibody. Underlined residues show discrepancies. “X” indicates aresidue missing from the published sequence. The correct amino acidsequences of murine IMMU-H3 heavy chain (SEQ ID NO:108) and light chain(SEQ ID NO:109) are shown alongside the incorrect published sequences ofmurine LG2-1 heavy chain (SEQ ID NO:122) and light chain (SEQ ID NO:123)

FIG. 3. Comparison of the variable region amino acid sequences of themurine IMMU-H2B heavy and light chains with the corresponding sequencespublished in Monestier et al. (1993, Mol. Immunol 30:1069-75) for theLG2-2 antibody. Underlined residues show discrepancies. “X” indicates aresidue missing from the published sequence. The correct amino acidsequences of murine IMMU-H2B heavy chain (SEQ ID NO:118) and light chain(SEQ ID NO:119) are shown alongside the incorrect published sequences ofmurine LG2-2 heavy chain (SEQ ID NO:124) and light chain (SEQ ID NO:125)

FIG. 4. Amino acid sequences of the heavy chain (SEQ ID NO:96) and lightchain (SEQ ID NO:97) variable regions of the humanized IMMU-H4 antibody.Residues that differ from the murine parent antibody are underlined.

FIG. 5. Amino acid sequences of the heavy chain (SEQ ID NO:106) andlight chain (SEQ ID NO:107) variable regions of the humanized IMMU-H3antibody. Residues that differ from the murine parent antibody areunderlined.

FIG. 6. Amino acid sequences of the heavy chain (SEQ ID NO:116) andlight chain (SEQ ID NO:117) variable regions of the humanized IMMU-H2Bantibody. Residues that differ from the murine parent antibody areunderlined.

FIG. 7. Comparative binding affinities of murine (squares) and chimeric(circles) IMMU-H4 anti-histone antibodies.

DEFINITIONS

Unless otherwise specified, “a” or “an” means “one or more”.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, peptides, drugs, toxins, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MRI).

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody). An “antibody” includes monoclonal, polyclonal, bispecific,multispecific, murine, chimeric, humanized and human antibodies.

A “naked antibody” is an antibody or antigen binding fragment thereofthat is not attached to a therapeutic or diagnostic agent. The Fcportion of an intact naked antibody can provide effector functions, suchas complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev50:59-87, 1998). Other mechanisms by which naked antibodies induce celldeath may include apoptosis. (Vaswani and Hamilton, Ann Allergy AsthmaImmunol 81: 105-119, 1998.)

An “antibody fragment” is a portion of an intact antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv, scFv, dAb and the like. Regardlessof structure, an antibody fragment binds with the same antigen that isrecognized by the full-length antibody. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains or recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). “Single-chain antibodies”, often abbreviated as“scFv” consist of a polypeptide chain that comprises both a V_(H) and aV_(L) domain which interact to form an antigen-binding site. The V_(H)and V_(L) domains are usually linked by a peptide of 1 to 25 amino acidresidues. Antibody fragments also include diabodies, triabodies andsingle domain antibodies (dAb). Fragments of antibodies that do not bindto the same antigen as the intact antibody, such as the Fc fragment, arenot included within the scope of an “antibody fragment” as used herein.

An anti-histone antibody or antibody fragment, or a compositiondescribed herein, is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient subject.In particular embodiments, an antibody preparation is physiologicallysignificant if its presence invokes an antitumor response or mitigatesthe signs and symptoms of an autoimmune disease state. A physiologicallysignificant effect could also be the evocation of a humoral and/orcellular immune response in the recipient subject leading to growthinhibition or death of target cells.

Anti-Histone Antibodies

Various humanized or chimeric anti-histone antibodies and/orantigen-binding fragments thereof are disclosed herein. The murine BWA-3(anti-H4), LG2-1 (anti-H3) and LG2-2 (anti-H2B) hybridomas from whichthe currently disclosed chimeric and humanized IMMU-H4, IMMU-H3 andIMMU-H2B antibodies were derived were reported by Monestier et al.(1993, Mol. Immunol 30:1069-75). However, murine antibodies aregenerally not appropriate for human therapeutic use, due to theformation of human anti-mouse antibodies (HAMA) that can neutralizethese anatibodies and thus make them less active. Further, the variableregion sequences reported by Monestier et al. (1993) for murine BWA-3,LG2-1 and LG2-2 were incorrect and/or incomplete and could not haveprovided the basis for production of chimeric or humanized antibodies.

In preferred embodiments, a humanized or chimeric IMMU-H4 antibody isone that comprises the heavy chain complementarity-determining region(CDR) sequences CDR1 (DDYLH, SEQ ID NO:90), CDR2 (WIG WIDPENGDTEYASKFQG,SEQ ID NO:91) and CDR3 (PLVHLRTFAY, SEQ ID NO:92) and the light chainCDR sequences CDR1 (RASESVDSYDNSLH, SEQ ID NO:93), CDR2 (LASNLES, SEQ IDNO:94) and CDR3 (QQNNEDPWT, SEQ ID NO:95). In more preferredembodiments, the humanized IMMU-H4 antibody comprises the heavy andlight chain variable region sequences of SEQ ID NO:96 and SEQ ID NO:97.In other preferred embodiments, the chimeric IMMU-H4 antibody comprisesthe heavy and light chain variable region sequences of SEQ ID NO:98 andSEQ ID NO:99.

Humanized IMMU-H4 VH Sequence (SEQ ID NO: 96)QVQLQQSGAEVKKPGSSVKVSCKASGYTFTDDYLHWVKQAPGQGLEWIGWIDPENGDTEYASKFQGKATLTADESTNTAYMELSSLRSEDTAFYYCARPL VHLRTFAYWGQGTTVTVSSHumanized IMMU-H4 VK Sequence (SEQ ID NO: 97)DIQLTQSPSSLSASVGDRVTMTCRASESVDSYDNSLHWFQQKPGKAPKPWIYLASNLESGVPVRFSGSGSGTDYTFTISSLQPEDIATYYCQQNNEDPWT FGGGTKLEIKRChimeric IMMU-H4 VH Sequence (SEQ ID NO: 98)QVQLQQSGAELVRPGASVKLSCTASGFNIKDDYLHWVKQRPEQGLEWIGWIDPENGDTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSSPL VHLRTFAYWGQGTLVTVSChimeric IMMU-H4 VK Sequence (SEQ ID NO: 99)DIQLTQSPASLAVSLGQRATISCRASESVDSYDNSLHWFQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWT FGGGTKLEIKR

In preferred embodiments, a humanized or chimeric IMMU-H3 antibody isone that comprises the heavy chain CDR sequences CDR1 (SYWMH, SEQ IDNO:100), CDR2 (NIDPSDSETHYNQKFKD, SEQ ID NO:101) and CDR3 (EKITDDYNYFDY,SEQ ID NO:102) and the light chain CDR sequences CDR1 (RASESVDSYGNSFMH,SEQ ID NO:103), CDR2 (HASNLES, SEQ ID NO:104) and CDR3 (QQNNEDPLT, SEQID NO:105). In more preferred embodiments, the humanized IMMU-H3antibody comprises the heavy and light chain variable region sequencesof SEQ ID NO:106 and SEQ ID NO:107. In other preferred embodiments, thechimeric IMMU-H3 antibody comprises the heavy and light chain variableregion sequences of SEQ ID NO:108 and SEQ ID NO:109.

Humanized IMMU-H3 VH Sequence (SEQ ID NO: 106)QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVKQAPGQGLEWIGNIDPSDSETHYNQKFKDKATLTADESTNTAYMELSSLRSEDTAFYYCAREK ITDDYNYFDYWGQGTTVTVSSHumanized IMMU-H3 VK Sequence (SEQ ID NO: 107)DIQLTQSPSSLSASVGDRVTMTCRASESVDSYGNSFMHWFQQKPGKAPKPWIYHASNLESGVPVRFSGSGSGTDYTFTISSLQPEDIATYYCQQNNEDPL TFGGGTKLEIKRChimeric IMMU-H3 VH Sequence (SEQ ID NO: 108)QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSNTAYMQLSSLTSEDSAVFYCAREK ITDDYNYFDYWGQGTTLTVSChimeric IMMU-H3 VK Sequence (SEQ ID NO: 109)DIQLTQSPASLALSLRQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYHASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPL TFGAGTKLELKR

In preferred embodiments, a humanized or chimeric IMMU-H2B antibody isone that comprises the heavy chain CDR sequences CDR1 (SYVMY, SEQ IDNO:110), CDR2 (YINPYNDGTKYNEKFKG, SEQ ID NO:111) and CDR3 (PGDGYPFDY,SEQ ID NO:112) and the light chain CDR sequences CDR1 (RSSQSIVHSNGNTYLE,SEQ ID NO:113), CDR2 (KVSNRFS, SEQ ID NO:114) and CDR3 (FQGSHVPYT, SEQID NO:115). In more preferred embodiments, the humanized IMMU-H2Bantibody comprises the heavy and light chain variable region sequencesof SEQ ID NO:116 and SEQ ID NO:117. In other preferred embodiments, thechimeric IMMU-H2B antibody comprises the heavy and light chain variableregion sequences of SEQ ID NO:118 and SEQ ID NO:119.

Humanized IMMU-H2B VH Sequence (SEQ ID NO: 116)QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYVMYWVKQAPGQGLEWIGYINPYNDGTKYNEKFKGKATLTADESTNTAYMELSSLRSEDTAFYYCARPG DGYPFDYWGQGTTVTVSSHumanized IMMU-H2B VK Sequence (SEQ ID NO: 117)DIQLTQSPSSLSASVGDRVTMTCRSSQSIVHSNGNTYLEWFQQKPGKAPKPWIYKVSNRFSGVPVRFSGSGSGTDYTFTISSLQPEDIATYYCFQGSHVP YTFGGGTKLEIKRChimeric IMMU-H2B VH Sequence (SEQ ID NO: 118)QVKLQQSGPELVKPGASVKMSCRASGYTFTSYVMYWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAGPG DGYPFDYWGQGTTLTVSChimeric IMMU-H2B VK Sequence (SEQ ID NO: 119)DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP YTFGSGTKLEIKR

General Techniques for Antibodies and Antibody Fragments

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XenoMouse® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

Techniques for producing single domain antibodies are also known in theart, as disclosed for example in Cossins et al. (2006, Prot ExpressPurif 51:253-259), incorporated herein by reference. Single domainantibodies (VHH) may be obtained, for example, from camels, alpacas orllamas by standard immunization techniques. (See, e.g., Muyldermans etal., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75,2003; Maass et al., J Immunol Methods 324:13-25, 2007). The VHH may havepotent antigen-binding capacity and can interact with novel epitopesthat are inacessible to conventional VH-VL pairs. (Muyldermans et al.,2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgGantibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized withknown antigens, such as TNF-α, and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). In certainembodiments, anti-pancreatic cancer VHH antibody fragments may beutilized in the claimed compositions and methods.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and/orhave published variable region sequences and are available for use inthe claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040; 6,451,310;6,444,206, 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Particular antibodies that may be of use for therapy of cancer withinthe scope of the claimed methods and compositions include, but are notlimited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7(anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX),hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan(anti-CD20); panitumumab (anti-EGFR); rituximab (anti-CD20); tositumomab(anti-CD20); GA101 (anti-CD20); and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20040202666(now abandoned); 20050271671; and 20060193865; the Examples section ofeach incorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab), CD74 (milatuzumab), CD19(hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see, e.g., Winer et al.,2011, Nature Med 17:610-18). Anti-CD3 antibodies also have been proposedfor therapy of type 1 diabetes (Cernea et al., 2010, Diabetes Metab Rev26:602-05).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

The pharmaceutical composition of the present invention may be used totreat a subject having a metabolic disease, such amyloidosis, or aneurodegenerative disease, such as Alzheimer's disease. Bapineuzumab isin clinical trials for Alzheimer's disease therapy. Other antibodiesproposed for therapy of Alzheimer's disease include Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition.

In a preferred embodiment, diseases that may be treated using theclaimed compositions and methods include cardiovascular diseases, suchas fibrin clots, atherosclerosis, myocardial ischemia and infarction.Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL), anti-MIF, and anti-CD74 (e.g., hLL1) antibodies canbe used to target atherosclerotic plaques. Abciximab (anti-glycoproteinIIb/IIIa) has been approved for adjuvant use for prevention ofrestenosis in percutaneous coronary interventions and the treatment ofunstable angina (Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3antibodies have been reported to reduce development and progression ofatherosclerosis (Steffens et al., 2006, Circulation 114:1977-84).Antibodies against oxidized LDL induced a regression of establishedatherosclerosis in a mouse model (Ginsberg, 2007, J Am Coll Cardiol52:2319-21). Anti-ICAM-1 antibody was shown to reduce ischemic celldamage after cerebral artery occlusion in rats (Zhang et al., 1994,Neurology 44:1747-51). Commercially available monoclonal antibodies toleukocyte antigens are represented by: OKT anti-T-cell monoclonalantibodies (available from Ortho Pharmaceutical Company) which bind tonormal T-lymphocytes; the monoclonal antibodies produced by thehybridomas having the ATCC accession numbers HB44, HB55, HB12, HB78 andHB2; G7E11, W8E7, NKP15 and GO22 (Becton Dickinson); NEN9.4 (New EnglandNuclear); and FMC11 (Sera Labs). A description of antibodies againstfibrin and platelet antigens is contained in Knight, Semin. Nucl. Med.,20:52-67 (1990).

Other antibodies that may be used include antibodies against infectiousdisease agents, such as bacteria, viruses, mycoplasma or otherpathogens. Many antibodies against such infectious agents are known inthe art and any such known antibody may be used in the claimed methodsand compositions. For example, antibodies against the gp120 glycoproteinantigen of human immunodeficiency virus I (HIV-1) are known, and certainof such antibodies can have an immunoprotective role in humans. See,e.g., Rossi et al., Proc. Natl. Acad. Sci. USA. 86:8055-8058, 1990.Known anti-HIV antibodies include the anti-envelope antibody describedby Johansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), as well as theanti-HIV antibodies described and sold by Polymun (Vienna, Austria),also described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agents Chemother. 2006; 50(5):1773-9, all incorporated herein byreference.

Antibodies against malaria parasites can be directed against thesporozoite, merozoite, schizont and gametocyte stages. Monoclonalantibodies have been generated against sporozoites (cirumsporozoiteantigen), and have been shown to neutralize sporozoites in vitro and inrodents (N. Yoshida et al., Science 207:71-73, 1980). Several groupshave developed antibodies to T. gondii, the protozoan parasite involvedin toxoplasmosis (Kasper et al., J. Immunol. 129:1694-1699, 1982; Id.,30:2407-2412, 1983). Antibodies have been developed againstschistosomular surface antigens and have been found to act againstschistosomulae in vivo or in vitro (Simpson et al., Parasitology,83:163-177, 1981; Smith et al., Parasitology, 84:83-91, 1982: Gryzch etal., J. Immunol., 129:2739-2743, 1982; Zodda et al., J. Immunol.129:2326-2328, 1982; Dissous et al., J. immunol., 129:2232-2234, 1982)

Trypanosoma cruzi is the causative agent of Chagas' disease, and istransmitted by blood-sucking reduviid insects. An antibody has beengenerated that specifically inhibits the differentiation of one form ofthe parasite to another (epimastigote to trypomastigote stage) in vitro,and which reacts with a cell-surface glycoprotein; however, this antigenis absent from the mammalian (bloodstream) forms of the parasite (Sheret al., Nature, 300:639-640, 1982).

Anti-fungal antibodies are known in the art, such as anti-Sclerotiniaantibody (U.S. Pat. No. 7,910,702); antiglucuronoxylomannan antibody(Zhong and Priofski, 1998, Clin Diag Lab Immunol 5:58-64); anti-Candidaantibodies (Matthews and Burnie, 2001, 2:472-76); andanti-glycosphingolipid antibodies (Toledo et al., 2010, BMC Microbiol10:47).

Suitable antibodies have been developed against most of themicroorganism (bacteria, viruses, protozoa, fungi, other parasites)responsible for the majority of infections in humans, and many have beenused previously for in vitro diagnostic purposes. These antibodies, andnewer antibodies that can be generated by conventional methods, areappropriate for use in the present invention.

Bispecific and Multispecific Antibodies

Bispecific or multispecific antibodies can be prepared by a variety ofprocedures, ranging from glutaraldehyde linkage to more specificlinkages between functional groups. The antibodies and/or antibodyfragments are preferably covalently bound to one another, directly orthrough a linker moiety, through one or more functional groups on theantibody or fragment, e. g., amine, carboxyl, phenyl, thiol, or hydroxylgroups. Various conventional linkers in addition to glutaraldehyde canbe used, e. g., disiocyanates, diiosothiocyanates,bis(hydroxysuccinimide) esters, carbodiimides,maleimidehydroxy-succinimde esters, and the like. The optimal length ofthe linker may vary according to the type of target cell.

A simple method to produce multivalent antibodies is to mix theantibodies or fragments in the presence of glutaraldehyde. The initialSchiff base linkages can be stabilized, e. g., by borohydride reductionto secondary amines. A diiosothiocyanate or carbodiimide can be used inplace of glutaraldehyde as a non-site-specific linker.

The simplest form of a multivalent, multispecific antibody is abispecific antibody. Bispecific antibodies can be made by a variety ofconventional methods, e. g., disulfide cleavage and reformation ofmixtures of whole IgG or, preferably F (ab′)₂ fragments, fusions of morethan one hybridoma to form polyomas that produce antibodies having morethan one specificity, and by genetic engineering. Bispecific antibodieshave been prepared by oxidative cleavage of Fab′ fragments resultingfrom reductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F (ab′)₂ fragments produced bypepsin digestion of two different antibodies, reductive cleavage to forma mixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F (ab′)₂ fragments includingbispecific antibodies containing a Fab′ portion specific to each of theoriginal epitopes.

General techniques for the preparation of multivalent antibodies may befound, for example, in Nisonhoff et al., Arch Biochem. Biophys, 93: 470(1961), Hammerling et al., J. Exp. Med. 128: 1461 (1968), and U.S. Pat.No. 4,331,647.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimide-hydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g., an antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e. g., Traut's Reagent. Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen binding sites. This can be accomplished by, e. g.,linkage to cleaved interchain sulfydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody which has at lease one free aminefunction. This results in an initial Schiff base (imine) linkage, whichis preferably stabilized by reduction to a secondary amine, e. g., byborohydride reduction, to form the final product. Such site-specificlinkages are disclosed, for small molecules, in U.S. Pat. No. 4,671,958,and for larger addends in U.S. Pat. No. 4,699,784.

Alternatively, such bispecific antibodies can be produced by fusing twohybridoma cell lines that produce appropriate Mabs. Techniques forproducing tetradomas are described, for example, by Milstein et al.,Nature 305: 537 (1983) and Pohl et al, Int. J. Cancer 54: 418 (1993).

Alternatively, chimeric genes can be designed that encode both bindingdomains. General techniques for producing bispecific antibodies bygenetic engineering are described, for example, by Songsivilai et al.,Biochem Biophys Res. Commun 164: 271 (1989); Trauneeker et al. EMBO J.10: 3655 (1991); and Weiner et al., J. Immunol. 147:4035 (1991).

A higher order multivalent, multispecific molecule can be obtained byadding various antibody components to a bispecific antibody, produced asabove. For example, a bispecific antibody can be reacted with2-iminothiolane to introduce one or more sulfhydryl groups for use incoupling the bispecific antibody to a further antibody derivative thatbinds an the same or a different epitope of the target antigen, usingthe bis-maleimide activation procedure described above. These techniquesfor producing multivalent antibodies are well known to those of skill inthe art. See, for example, U.S. Pat. No. 4,925,648, and Goldenberg,international publication No. WO 92/19273, which are incorporated byreference.

DOCK-AND-LOCK™ (DNL™)

In preferred embodiments, a bispecific or multispecific antibody isformed as a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos.7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examplessection of each of which is incorporated herein by reference.)Generally, the technique takes advantage of the specific andhigh-affinity binding interactions that occur between a dimerization anddocking domain (DDD) sequence of the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequencederived from any of a variety of AKAP proteins (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein,peptide or other molecule. Because the DDD sequences spontaneouslydimerize and bind to the AD sequence, the technique allows the formationof complexes between any selected molecules that may be attached to DDDor AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL™constructs of different stoichiometry may be produced and used (see,e.g., U.S. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL™construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA  AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC 

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ(SEQ ID NO: 9) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Can et al., 2001, J Biol Chem 276:17332-38; Altoet al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al.,2006, Biochem J396:297-306; Stokka et al., 2006, Biochem J400:493-99;Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408, the entire text of each of which is incorporated herein byreference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 1. In devising Table 1, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 1 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 87. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V V

(SEQ ID NO: 12) THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 13) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 14) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 15) SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 16) SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 17) SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 18) SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 19) SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 20) SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 21) SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 22) SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 23) SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 24) SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 25) SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA(SEQ ID NO: 26) SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA(SEQ ID NO: 27) SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA(SEQ ID NO: 28) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA(SEQ ID NO: 29) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA(SEQ ID NO: 30) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA(SEQ ID NO: 31) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 2 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 1 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 2 Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 3).Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y L A K Q I V D N AI Q Q A N L D F I R N E Q N N L V T V I S V

(SEQ ID NO: 32) NIEYLAKQIVDNAIQQA (SEQ ID NO: 33) QLEYLAKQIVDNAIQQA(SEQ ID NO: 34) QVEYLAKQIVDNAIQQA (SEQ ID NO: 35) QIDYLAKQIVDNAIQQA(SEQ ID NO: 36) QIEFLAKQIVDNAIQQA (SEQ ID NO: 37) QIETLAKQIVDNAIQQA(SEQ ID NO: 38) QIESLAKQIVDNAIQQA (SEQ ID NO: 39) QIEYIAKQIVDNAIQQA(SEQ ID NO: 40) QIEYVAKQIVDNAIQQA (SEQ ID NO: 41) QIEYLARQIVDNAIQQA(SEQ ID NO: 42) QIEYLAKNIVDNAIQQA (SEQ ID NO: 43) QIEYLAKQIVENAIQQA(SEQ ID NO: 44) QIEYLAKQIVDQAIQQA (SEQ ID NO: 45) QIEYLAKQIVDNAINQA(SEQ ID NO: 46) QIEYLAKQIVDNAIQNA (SEQ ID NO: 47) QIEYLAKQIVDNAIQQL(SEQ ID NO: 48) QIEYLAKQIVDNAIQQI (SEQ ID NO: 49) QIEYLAKQIVDNAIQQV

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIa. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIa wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA(SEQ ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 55) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 57)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 59) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 63) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 3 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 3 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Can et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIa DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 4. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 1 andTable 2.

TABLE 4 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 89. S H I Q I P P G L T E L LQ G Y T V E V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A RA N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Alternative DNL™ Structures

In certain alternative embodiments, DNL™ constructs may be formed usingalternatively constructed antibodies or antibody fragments, in which anAD moiety may be attached at the C-terminal end of the kappa light chain(C_(k)), instead of the C-terminal end of the Fc on the heavy chain. Thealternatively formed DNL™ constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. Nos. 61/654,310, filed Jun. 1,2012, 61/662,086, filed Jun. 20, 2012, 61/673,553, filed Jul. 19, 2012,and 61/682,531, filed Aug. 13, 2012, the entire text of eachincorporated herein by reference. The light chain conjugated DNL™constructs exhibit enhanced Fc-effector function activity in vitro andimproved pharmacokinetics, stability and anti-lymphoma activity in vivo(Rossi et al., 2013, Bioconjug Chem 24:63-71).

C_(k)-conjugated DNL™ constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. Nos. 61/654,310, 61/662,086,61/673,553, and 61/682,531. Briefly, C_(k)-AD2-IgG, was generated byrecombinant engineering, whereby the AD2 peptide was fused to theC-terminal end of the kappa light chain. Because the natural C-terminusof C_(K) is a cysteine residue, which forms a disulfide bridge toC_(H)1, a 16-amino acid residue “hinge” linker was used to space the AD2from the C_(K)-V_(H)1 disulfide bridge. The mammalian expression vectorsfor C_(k)-AD2-IgG-veltuzumab and C_(k)-AD2-IgG-epratuzumab wereconstructed using the pdHL2 vector, which was used previously forexpression of the homologous C_(H)3-AD2-IgG modules. A 2208-bpnucleotide sequence was synthesized comprising the pdHL2 vector sequenceranging from the Bam HI restriction site within the V_(K)/C_(K) intronto the Xho I restriction site 3′ of the C_(k) intron, with the insertionof the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ IDNO:126) and AD2, in frame at the 3′end of the coding sequence for C_(K).This synthetic sequence was inserted into the IgG-pdHL2 expressionvectors for veltuzumab and epratuzumab via Bam HI and Xho I restrictionsites. Generation of production clones with SpESFX-10 were performed asdescribed for the C_(H)3-AD2-IgG modules. C_(k)-AD2-IgG-veltuzumab andC_(k)-AD2-IgG-epratuzumab were produced by stably-transfected productionclones in batch roller bottle culture, and purified from the supernatantfluid in a single step using MabSelect (GE Healthcare) Protein Aaffinity chromatography.

Following the same DNL process described previously for 22-(20)-(20)(Rossi et al., 2009, Blood 113:6161-71), C_(k)-AD2-IgG-epratuzumab wasconjugated with C_(H)1-DDD2-Fab-veltuzumab, a Fab-based module derivedfrom veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the 22*indicates the C_(k)-AD2 module of epratuzumab and each (20) symbolizes astabilized dimer of veltuzumab Fab. The properties of 22*-(20)-(20) werecompared with those of 22-(20)-(20), the homologous Fc-bsHexAbcomprising C_(H)3-AD2-IgG-epratuzumab, which has similar composition andmolecular size, but a different architecture.

Following the same DNL process described previously for 20-2b (Rossi etal., 2009, Blood 114:3864-71), C_(k)-AD2-IgG-veltuzumab, was conjugatedwith IFNα2b-DDD2, a module of IFNα2b with a DDD2 peptide fused at itsC-terminal end, to generate 20*-2b, which comprises veltuzumab with adimeric IFNα2b fused to each light chain. The properties of 20*-2b werecompared with those of 20-2b, which is the homologous Fc-IgG-IFNα.

Each of the bsHexAbs and IgG-IFNα were isolated from the DNL reactionmixture by MabSelect affinity chromatography. The two C_(k)-derivedprototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprisingepratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and aCD20-targeting immunocytokine, comprising veltuzumab and four moleculesof interferon-α2b, displayed enhanced Fc-effector functions in vitro, aswell as improved pharmacokinetics, stability and anti-lymphoma activityin vivo, compared to their Fc-derived counterparts.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab (SEQ IDNO:86).

Rituximab heavy chain variable region sequence (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 86) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotypoe characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Exemplary antibody constant region sequences of use in the chimeric andhumanized anti-histone antibodies are disclosed in SEQ ID NO:127 and SEQID NO:128 below.

Exemplary human heavy chain constant region (SEQ ID NO: 127)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Exemplary human light chain constant region(SEQ ID NO: 128) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

Immunoconjugates

In certain embodiments, the antibodies or fragments thereof may beconjugated to one or more therapeutic or diagnostic agents. Thetherapeutic agents do not need to be the same but can be different, e.g.a drug and a radioisotope. For example, ¹³¹I can be incorporated into atyrosine of an antibody or fusion protein and a drug attached to anepsilon amino group of a lysine residue. Therapeutic and diagnosticagents also can be attached, for example to reduced SH groups and/or tocarbohydrate side chains. Many methods for making covalent ornon-covalent conjugates of therapeutic or diagnostic agents withantibodies or fusion proteins are known in the art and any such knownmethod may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.Pat. No. 7,563,433, the Examples section of which is incorporated hereinby reference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MRI. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F-Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

Therapeutic Agents

In alternative embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes orother agents may be used, either conjugated to the subject anti-histoneantibodies or separately administered before, simultaneously with, orafter the anti-histone antibody. Drugs of use may possess apharmaceutical property selected from the group consisting ofantimitotic, antikinase (e.g., anti-tyrosine kinase), alkylating,antimetabolite, antibiotic, alkaloid, anti-angiogenic, pro-apoptoticagents, immune modulators, and combinations thereof.

Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine,anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-B, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. Lenolidamide is yet another immunomodulator that has shownactivity in controlling certain cancers, such as multiple myeloma andhematopoietic tumors.

Radionuclides of use include, but are not limited to—¹¹¹In, ¹⁷⁷Ln,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb and ²²⁷Th. The therapeuticradionuclide preferably has a decay-energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227, and Fm-255. Decayenergies of useful alpha-particle-emitting radionuclides are preferably2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably4,000-7,000 keV. Additional potential radioisotopes of use include ¹¹C,¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru,¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm,¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au,⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.Some useful diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or, ¹¹¹In.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2. A preferred formof therapeutic oligonucleotide is siRNA.

Diagnostic Agents

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹¹⁰In,¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴-¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III). Ultrasound contrast agents may comprise liposomes, suchas gas filled liposomes. Radiopaque diagnostic agents may be selectedfrom compounds, barium compounds, gallium compounds, and thalliumcompounds. A wide variety of fluorescent labels are known in the art,including but not limited to fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. Chemiluminescent labels of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt or an oxalate ester.

Immune Dysregulatory Disease, Infectious Disease and InflammatoryDisease

In various embodiments, the anti-histone antibodies or fragments thereofare of use to treat inflammatory or immune-dysregulatory diseases, suchas sepsis, septic shock, septicemia, acute respiratory distresssyndrome, graft-vs.host disease (GVHD), transplant rejection,atherosclerosis, asthma, granulomatous disease, a neuropathy, cachexia,a coagulopathy, acne, giant cell arteritis or myocardial ischemia, aswell as typical autoimmune disease (as listed previously). In certainpreferred embodiments, the therapy may utilize either a combination oftwo or more separate antibodies or fragments thereof, administeredtogether or separately, or else a bispecific or multispecific antibodyor antibody fragment, with a first binding site for a histone and asecond binding site for a different target antigen. More preferably, atarget antigen may be selected from the group consisting of histone H3,histone H4, histone H2B, a proinflammatory effector of the innate immunesystem, a component or cell of the adaptive immune system, aproinflammatory effector cytokine or chemokine, or a target specificallyassociated with infectious disease, acute respiratory distress syndrome,septicemia, septic shock, GVHD, transplant rejection, atherosclerosis,asthma, granulomatous disease, a neuropathy, cachexia, a coagulopathy,acne, giant cell arteritis or myocardial ischemia. In some cases, CD74may be specifically excluded as a potential target antigen, except whenthe anti-CD74 antibody or inhibitor is combined with an anti-histoneantibody. Specific target antigens of use may include, but are notlimited to, TNF-α, MIF, CD74, HLA-DR, IL-1, IL-3, IL-4, IL-5, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, CD40L, CD44, CD46, CD55, CD59, CCL19, CCL21, mCRP,MCP-19, MIP-1A, MIP-1B, RANTES, ENA-78, IP-10, GRO-β,lipopolysaccharide, lymphotoxin, HMGB-1, tissue factor, a complementregulatory protein, a coagulation factor, thrombin, a complement factor,C3, C3a, C3b, C4a, C4b, C5, C5a, C5b, Flt-1 and VEGF. Thrombomodulinand/or activated protein C may also be combined with anti-histoneantibodies used with any of the above-specified antibodies.

Additional therapeutic agents that may be added in combination include acytokine, a chemokine, a coagulation inhibitor, an anti-T cell or antiB-cell antibody or antibody fragment, an immunomodulator, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor, an interferon, erythropoietin or thrombopoietin. Anoptional therapeutic agent may include activated protein C orthrombomodulin, as mentioned above.

Embodiments of the invention relate generally to methods andcompositions for immunotherapy of inflammatory and immune-dysregulatorydiseases, using multispecific antibodies that target at least twodifferent markers. The markers may be antigens and/or receptors onlymphocytes, macrophages, monocytes, or dendritic cells (DCs).Particular embodiments relate to methods and compositions for modulatingreceptors on immune-targeting and immune-processing cells using specificantibodies and antibody heteroconjugates to bind to the cells and theirreceptors, to effect a treatment of various diseases that are generatedor exacerbated by, or otherwise involve, these cells and theirreceptors. Such diseases more particularly include acute and chronicinflammatory disorders, autoimmune diseases, septicemia and septicshock, neuropathies, graft-versus-host disease, acute respiratorydistress syndrome, granulomatous diseases, giant cell arteritis, acne,diffuse intravascular coagulation (DIC), transplant rejection, asthma,cachexia, myocardial ischemia, and atherosclerosis. The methods andcompositions also are useful in treating pathological angiogenesis andcancer. The methods and compositions can include a secondary therapeuticthat is directed to a cancer receptor, a cancer oncogene, orcancer-associated antigen. Methods and compositions are also describedfor improved diagnosis/detection of the diseases.

BACKGROUND

The immune system comprises both the innate immune system and theadaptive, or acquired immune system. Many host cells participate in theprocesses of innate and adaptive immunity, such as neutrophils, T- andB-lymphocytes, macrophages and monocytes, dendritic cells, and plasmacells. They usually act in concert, affecting one another, particularlyin the regulation of certain factors and cytokines that contribute tothe recognition and processing of innate and external noxients, andthese systems have evolved over the millions of years of the developmentof vertebrate, mammalian, and human organisms.

A major goal of immunotherapy is to exploit or enhance a patient'simmune system against an innate or foreign noxient, such as a malignantcell or an invading microorganism. The immune system has been studiedmore in relation to recognizing and responding to exogenous noxients,such as microbial organisms, than it has in relation to indigenousmalfunctions, such as cancer and certain autoimmune andimmune-dysregulatory diseases, particularly since the latter may haveboth genetic as well as environmental components. The defenses againstmicrobial organisms, such as bacteria, fungi, parasites, and viruses,are innate to the particular organism, with the immune system beingprogrammed to recognize biochemical patterns of these microorganisms andto respond to attack them without requiring prior exposure to themicroorganism. This innate immune system includes, for example,neutrophils, natural killer cells and monocytes/macrophages that caneradicate the invading microorganisms by direct engulfment anddestruction.

The innate immune response is often referred to as a nonspecific onethat controls an invading external noxient until the more specificadaptive immune system can marshal specific antibodies and T cells (cf.Modlin et al., N Engl J Med 1999, 340:1834-1835; Das, Crit. Care 2000;4:290-296). The nonspecific immune responses involve the lymphaticsystem and phagocytes. The lymphatic system includes the lymphocytes andmacrophages. Macrophages can engulf, kill and dispose of foreignparticles. Phagocytes include neutrophils and macrophages, which againingest, degrade and dispose of debris, and have receptors for complementand antibody. In summary, the innate immune system provides a line ofdefense again certain antigens because of inherited characteristics.

In contrast, the adaptive, or acquired, immune system, is highly evolvedand very specific in its responses. It is called an adaptive systembecause is occurs during the lifetime of an individual as an adaptationto infection with a pathogen. Adaptive immunity can be artificiallyacquired in response to a vaccine (antigens) or by administeringantibodies, or can be naturally acquired by infection. The acquiredimmunity can be active, if an antibody was produced, or it can bepassive, if exogenous antibody made form another source is injected.

The adaptive immune system produces antibodies specific to a givenantigen. The simplest and most direct way in which antibodies provideprotection is by binding to them and thereby blocking their access tocells that they may infect or destroy. This is known as neutralization.Binding by antibodies, however, is not sufficient to arrest thereplication of bacteria that multiply outside cells. In this case, onerole of antibody is to enable a phagocytic cell to ingest and destroythe bacterium. This is known as opsonization. The third function ofantibodies is to activate a system of plasma proteins, known ascomplement. In many cases, the adaptive immune system confers lifelongprotective immunity to re-infection with the same pathogen, because theadaptive immune system has a ‘memory’ of the antigens presented to it.

Antibody-mediated immunity is called humoral immunity and is regulatedby B cells and the antibodies they produce. Cell-mediated immunity iscontrolled by T cells. Both humoral and cell-mediated immunityparticipate in protecting the host from invading organisms. Thisinterplay can result in an effective killing or control of foreignorganisms. Occasionally, however, the interplay can become erratic. Inthese cases, there is a dysregulation that can cause disease. Sometimesthe disease is life-threatening, such as with septic shock and certainautoimmune disorders.

The B and T lymphocytes are critical components of a specific immuneresponse. B cells are activated by antigen to engender clones ofantigen-specific cells that mediate adaptive immunity. Most clonesdifferentiate to plasma cells that secrete antibody, while a few clonesform memory cells that revert to plasma cells. Upon subsequentre-infection, memory cells produce a higher level of antibody in ashorter period than in the primary response. Antibodies secreted by theplasma cells can play multiple roles in immunity, such as binding andneutralizing a foreign agent, acting as opsonins (IgG) to promotephagocytosis, directly affecting metabolism and growth of someorganisms, engaging in antigen-antibody reactions that activatecomplement, causing phagocytosis and membrane attack complex, and/orengaging in antigen-antibody reactions that activate T cells and otherkiller cells.

T lymphocytes function as both helper cells and suppressor cells. HelperT cells induce antigen-specific B cells and effector T cells toproliferate and differentiate. Suppressor T cells interact with helper Tcells to prevent an immune response or to suppress an ongoing one, or toregulate effector T cells. Cytotoxic T cells destroy antigen by bindingto target cells. In a delayed-type hypersensitivity reaction, the Tcells do not destroy antigen, but attract macrophages, neutrophils andother cells to destroy and dispose of the antigen.

T cells can detect the presence of intracellular pathogens becauseinfected cells display on their surface peptide fragments derived fromthe pathogens' proteins. These foreign peptides are delivered to thecell surface by specialized host-cell glycoproteins, termed MajorHistocompatibility Complex (MHC) molecules. The recognition of antigenas a small peptide fragment bound to a MHC molecule and displayed at thecell surface is one of the most distinctive features of T cells. Thereare two different classes of MHC molecules, know as MHC class I and MHCclass II, that deliver peptides from different cellular compartments tothe surface of the infected cell. Peptides from the cytosol are bound toMHC class I molecules which are expressed on the majority of nucleatedcells and are recognized by CD8+ T cells. MHC class II molecules, incontrast, traffic to lysosomes for sampling endocytosed protein antigenswhich are presented to the CD4+ T cells (Bryant and Ploegh, Curr OpinImmunol 2004; 16:96-102).

CD8+ T cells differentiate into cytotoxic T cells, and kill the cell.CD4+ T cells differentiate into two types of effector T cells. Pathogensthat accumulate in large numbers inside macrophage vesicles tend tostimulate the differentiation of T_(H1) cells which activate macrophagesand induce B cells to make IgG antibodies that are effective inopsonizing extracellular pathogens for uptake by phagocytes.Extracellular antigens tend to stimulate the production of T_(H2) cellswhich initiate the humoral immune response by activating naiveantigen-specific B cells to produce IgM antibodies, inter alia.

The innate and adaptive immune systems interact, in that the cells ofthe innate immune system can express various molecules that can interactwith or trigger the adaptive immune system by activating certain cellscapable of producing immune factors, such as by activating T and B cellsof the lymphatic series of leukocytes. The early induced butnon-adaptive responses are important for two main reasons. First, theycan repel a pathogen or, more often, control it until an adaptive immuneresponse can be mounted. Second, these early responses influence theadaptive response in several ways. For example, the innate immuneresponse produces cytokines and other inflammatory mediators that haveprofound effects on subsequent events, including the recruitment of newphagocytic cells to local sites of infection. Another effect of thesemediators is to induce the expression of adhesion molecules on theendothelial cells of the local blood vessels, which bind to the surfaceof circulating monocytes and neutrophils and greatly increase their rateof migration of these cells out of the blood and into the tissues. Theseevents all are included under the term inflammation, which is a featureof the innate immune system that forms part of the protective responseat a localized site to isolate, destroy and remove a foreign material.This is followed by repair. Inflammation is divided into acute andchronic forms.

The immune system communicates via nonspecific tissue resistancefactors. These include the interferons, which are proteins produced inresponse to viruses, endotoxins and certain bacteria. Interferonsinhibit viral replication and activate certain host-defense responses.Infected cells produce interferon that binds the infected cells toother, neighboring cells, causing them to produce antiviral proteins andenzymes that interfere with viral gene transcription and proteinssynthesis. Interferons can also affect normal cell growth and suppresscell-mediated immunity.

Complement is another nonspecific tissue resistance factor, andcomprises plasma proteins and membrane proteins that mediate specificand non-specific defenses. Complement has two pathways, the classicalpathway associated with specific defense, and the alternative pathwaythat is activated in the absence of specific antibody, and is thusnon-specific. In the classical pathway, antigen-antibody complexes arerecognized when C1 interacts with the Fc of the antibody, such as IgMand to some extent, IgG, ultimately causing mast cells to releasechemotactic factors, vascular mediators and a respiratory burst inphagocytes, as one of many mechanisms. The key complement factorsinclude C3a and C5a, which cause mast cells to release chemotacticfactors such as histamine and serotonin that attract phagocytes,antibodies and complement, etc. Other key complement factors are C3b andC5b, which enhance phagocytosis of foreign cells, and C8 and C9, whichinduce lysis of foreign cells (membrane attack complex).

Cancer cells can escape immune surveillance by avoiding complementactivation, especially by the expression of membrane-associatedcomplement regulatory proteins, such as CD55 (decay-acceleratingfactor), CD46 (membrane cofactor protein), and CD59 (protectin), and itis believed that the over-expression of these proteins on cancer cellmembranes protects these cancers from complement activation (Brasoveanuet al., Lab Invest 1996; 74:33-42; Jarvis et al., Int J Cancer 1997;71:1049-1055; Yu et al., Clin Exp Immunol 1999; 115:13-18; Murray etal., Gynecol Oncol 2000; 76:176-182; Donin et al., Clin Exp Immunol2003; 131:254-263). Attempts have been made, unsuccessfully, to increasethe susceptibility to complement-mediated lysis by use of neutralizingantibodies against CD46, CD55 and CD59 (Varsano et al., Clin Exp Immunol1998; 113:173-182 Junnikkala et al., J Immunol 2000; 164:6075-6081;Maenpaa et al., Am J Pathol 1996; 148:1139-1162; Goiter, Lab Invest1996; 74:1039-1049. In the latter study, CD46 and CD55 antibodies were,in contrast to CD59 antibodies, ineffective. This suggests that othertargets, or the use of antibodies against multiple complement regulatoryproteins, or against both complement regulatory proteins and othermediators of immunity may be required. This general failure contradictsthe speculation of Fishelson et al. (Mol Immunol 2003: 40:109-123) andthe suggestion from other studies that treatment of cancer patients withantibodies to membrane complement regulatory proteins in combinationwith anticancer complement-fixing antibodies will improve therapeuticefficacy.

Gelderman et al. (Mol Immunol 2003; 40:13-23) reported thatmembrane-bound complement regulatory proteins (mCRP) inhibit complementactivation by an immunotherapeutic mAb in a syngeneic rat colorectalcancer model. While the use of mAb against tumor antigens and mCRPovercame an observed effect of mCRP on tumor cells, there has been nodirect evidence to support this approach. Still other attempts to usebispecific antibodies against CD55 and against a tumor antigen (G250 orEpCAM) have been suggested by Gelderman et al. (Lab Invest 2002;82:483-493; Eur J Immunol 2002; 32:128-135) based on in vitro studiesthat showed a 2-13-fold increase in C3 deposition compared to use of theparental antitumor antibody. However, no results involving enhanced cellkilling were reported. Jurianz et al. (Immunopharmacology 1999;42:209-218) also suggested that combining treatment of a tumor withanti-HER2 antibodies in vitro could be enhanced by prior treatment withantibody-neutralization of membrane-complement-regulatory protein, butagain no in vivo results were provided. Sier et al. (Int J Cancer 2004;109:900-908) recently reported that a bispecific antibody made againstan antigen expressed on renal cell carcinoma (Mab G250) and CD55enhanced killing of renal cancer cells in spheroids when beta-glucan wasadded, suggesting that the presence of CR3-priming beta-glucan wasobligatory.

Neutrophils, another cell involved in innate immune response, alsoingest, degrade and dispose of debris. Neutrophils have receptors forcomplement and antibody. By means of complement-receptor bridges andantibody, the foreign noxients can be captured and presented tophagocytes for engulfment and killing.

Macrophages are white blood cells that are part of the innate systemthat continually search for foreign antigenic substances. As part of theinnate immune response, macrophages engulf, kill and dispose of foreignparticles. However, they also process antigens for presentation to B andT cells, invoking humoral or cell-mediated immune responses.

The dendritic cell is one of the major means by which innate andadaptive immune systems communicate (Reis e Sousa, Curr Opin Immunol2004; 16:21-25). It is believed that these cells shape the adaptiveimmune response by the reactions to microbial molecules or signals.Dendritic cells capture, process and present antigens, thus activatingCD4+ and CD8+ naive T lymphocytes, leading to the induction of primaryimmune responses, and derive their stimulatory potency from expressionof MHC class I, MHC class II, and accessory molecules, such as CD40,CD54, CD80, CD86, and T-cell activating cytokines (Steinman, J ExpHematol 1996; 24:859-862; Banchereau and Steinman, Nature 1998;392:245-252). These properties have made dendritic cells candidates forimmunotherapy of cancers and infectious diseases (Nestle, Oncogene 2000;19:673-679; Fong and Engleman, Annu Rev Immunol 2000; 18:245-273;Lindquist and Pisa, Med Oncol 2002; 19:197-211), and have been shown toinduce antigen-specific cytotoxic T cells that result in strong immunityto viruses and tumors (Kono et al., Clin Cancer Res 2002; 8:394-40).

Also important for interaction of the innate and adaptive immune systemsis the NK cell, which appears as a lymphocyte but behaves like a part ofthe innate immune system. NK cells have been implicated in the killingof tumor cells as well as essential in the response to viral infections(Lanier, Curr Opin Immunol 2003; 15:308-314; Carayannopoulos andYokoyama, Curr Opin Immunol 2004; 16:26-33). Yet another importantmechanism of the innate immune system is the activation of cytokinemediators that alert other cells of the mammalian host to the presenceof infection, of which a key component is the transcription factor NF-κB(Li and Verna, Nat Rev Immunol 2002; 2:725-734).

As mentioned earlier, the immune system can overreact, resulting inallergies or autoimmune diseases. It can also be suppressed, absent, ordestroyed, resulting in disease and death. When the immune system cannotdistinguish between “self” and “nonself,” it can attach and destroycells and tissues of the body, producing autoimmune diseases, e.g.,juvenile diabetes, multiple sclerosis, myasthenia gravis, systemic lupuserythematosus, rheumatoid arthritis, and immune thrombocytopenicpurpura. Immunodeficiency disease results from the lack or failure ofone or more parts of the immune system, and makes the individualssusceptible to diseases that usually do not affect individuals with anormal immune system. Examples of immunodeficiency disease are severecombined immunodeficiency disease (SCID) and acquired immunodeficiencydisease (AIDS). The latter results from human immunodeficiency virus(HIV) and the former from enzyme or other inherited defects, such asadenosine deaminase deficiency.

The application of immunotherapy to cancer involves a number ofapproaches to engage or exploit the immune system, such as adoptivetransfer of anti-tumor-reactive T cells and the use of vaccines, as wellas breaking tolerance to tumor self-antigens by inhibiting regulatorycells, and boosting T-cell immunity by use of various cytokines andso-called immune-enhancing molecules (Antonia et al., Curr Opin Immunol2004; 16:130-136). Dendritic-cell vaccines have also been described.Direct and indirect (mediated by host effector cells) actions ofantibodies administered to patients by targeting tumor-cellantigens/receptors have now entered the cancer therapy armamentarium, asexemplified by antibodies against CD20 and CD52 in the therapy oflymphomas and leukemia; anti-epidermal growth factor receptor (EGFR),the anti-HER2/neu variant, in the therapy of diverse solid tumors; andanti-vascular endothelium growth factor (VEGF) for the treatment ofcertain solid tumors. Although active when given alone, most of theseshow enhanced antitumor effects when combined with other treatmentmodalities, such as drugs and radiation. Using these tumor-targetingantibodies to deliver cytotoxic drugs or isotopes is still anothermethod of immunotherapy that has entered the clinic. These and othermethods of cancer immunotherapy have been reviewed in Huber and Wolfel,J Cancer Res Clin Oncol 2004; 130:367-374. However, at best theseapproaches show reduction of tumor and improved survival in a proportionof the patients, most of whom eventually relapse, thus requiring othertherapeutic interventions and different strategies to control theirdisease.

Sepsis is a major medical and economic burden to our society, affectingabout 700,000 people annually in the United States, causing over 200,000deaths annually, and costing approximately $16.7 billion per year (Anguset al., Crit Care Med 2001; 29:1303-1310; Martin et al., N Engl J Med2003; 348:1546-1554). The definition of sepsis has been difficult, andhistorically it was defined as the systemic host response to aninfection. A discussion of the clinical definition of sepsis,encompassing systemic inflammatory response syndrome (SIRS), sepsis perse, severe sepsis, septic shock, and multiple organ dysfunction syndrome(MODS) is contained in Riedmann et al., J Clin Invest 2003; 112:460-467.Since it has been a common belief that sepsis is caused by the host'soverwhelming reaction to the invading microorganisms, and that thepatient is more endangered by this response that than the invadingmicroorganisms, suppression of the immune and inflammatory responses wasan early goal of therapy.

Numerous and diverse methods of immunosuppression or of neutralizingproinflammatory cytokines have proven to be unsuccessful clinically inpatients with sepsis and septic shock anti-inflammatory strategies.(Riedmann, et al., cited above; Van Amersfoort et al. (Clin MicrobiolRev 2003; 16:379-414), such as general immunosuppression, use ofnonsteroidal anti-inflammatory drugs, TNF-α antibody (infliximab) or aTNF-R:Fc fusion protein (etanercept), IL-1 (interleukin-1) receptorantagonist, or high doses of corticosteroids. However, a success in thetreatment of sepsis in adults was the PROWESS study (Human ActivatedProtein C Worldwide Evaluation in Severe Sepsis (Bernard et al., N EnglJ Med 2001; 344:699-709)), showing a lower mortality (24.7%) than in theplacebo group (30.8%). This activated protein C (APC) agent probablyinhibits both thrombosis and inflammation, whereas fibrinolysis isfostered. Friggeri et al. (2012, Mol Med 18:825-33) reported that APCdegrades histones H3 and H4, which block uptake and clearance ofapoptotic cells by macrophages and thereby contribute to organ systemdysfunction and mortality in acute inflammatory states. Van Amersfoortet al. state, in their review (ibid.) that: “Although the blocking ormodulation of a number of other targets including complement andcoagulation factors, neutrophil adherence, and NO release, are promisingin animals, it remains to be determined whether these therapeuticapproaches will be effective in humans.” This is further emphasized in areview by Abraham, “Why immunomodulatory therapies have not worked insepsis” (Intensive Care Med 1999; 25:556-566). In general, although manyrodent models of inflammation and sepsis have shown encouraging resultswith diverse agents over the past decade or more, most agents translatedto the clinic failed to reproduce in humans what was observed in theseanimal models, so that there remains a need to provide new agents thatcan control the complex presentations and multiple-organ involvement ofvarious diseases involving sepsis, coagulopathy, and certainneurodegenerative conditions having inflammatory or immune dysregulatorycomponents.

More recent work on immunoglobulins in sepsis or septic shock has beenreported. For example, Toussaint and Gerlach (2012, Curr Infect Dis Rep14:522-29) summarized the use of ivIG as an adjunct therapy in sepsis.The metanalysis failed to show any strong correlation between generalimmunoglobulin therapy and outcome. LaRosa and Opal (2012, Curr InfectDis Rep 14:474-83) reported on new therapeutic agents of potential usein sepsis. Among other agents, anti-TNF antibodies are in currentclinical trials for sepsis, while complement antagonists have shownpromising results in preclinical models of sepsis. Nalesso et al. (2012,Curr Infect Dis Rep 14:462-73) suggested that combination therapies withmultiple agents may prove more effective for sepsis treatment. Theimmunopathogenesis of sepsis has been summarized by Cohen (2002, Nature420:885-91).

The immune system in sepsis is believed to have an early intenseproinflammatory response after infection or trauma, leading to organdamage, but it is also believed that the innate immune system oftenfails to effectively kill invading microorganisms (Riedmann and Ward,Expert Opin Biol Ther 2003; 3:339-350). There have been some studies ofmacrophage migration inhibitory factor (MIF) in connection with sepsisthat have shown some promise. For example, blockage of MIF or targeteddisruption of the MIF gene significantly improved survival in a model ofseptic shock in mice (Calandra et al., Nature Med 2000; 6:164-170), andseveral lines of evidence have pointed to MIF as a potential target fortherapeutic intervention in septic patients (Riedmann et al., citedabove). Bucala et al. (U.S. Pat. No. 6,645,493 B1) have claimed that ananti-MIF antibody can be effective therapeutically for treating acondition or disease caused by cytokine-mediated toxicity, includingdifferent forms of sepsis, inflammatory diseases, acute respiratorydisease syndrome, granulomatous diseases, chronic infections, transplantrejection, cachexia, asthma, viral infections, parasitic infections,malaria, and bacterial infections, which is incorporated herein in itsentirety, including references. The use of anti-LPS (lipopolysaccharide)antibodies alone similarly has had mixed results in the treatment ofpatients with septic shock (Astiz and Rackow, Lancet 1998;351:1501-1505; Van Amersfoort et al., Clin Microbiol Rev 2003;16:379-414.

While both LPS and MIF have been pursued as targets in the treatment ofsepsis and septic shock, approaches which target LPS or MIF alone by anantibody have not been sufficient to control the diverse manifestationsof sepsis, especially in advanced and severe forms. Similarly, use ofcytokines, such as IL-1, IL-6 (interleukin-6), IL-8 (interleukin-8),etc., as targets for antibodies for the treatment of sepsis and othercytokine-mediated toxic reactions, has not proven to be sufficient for ameaningful control of this disease. Therefore, in addition to the needto discover additional targets of the cytokine cascade involved in theendogenous response in sepsis, it has now been discovered that bi- andmulti-functional antibodies targeting at least one cytokine or causativeagent, such as MIF or lipopolysaccharide (LPS), is advantageous,especially when combined with the binding to a host cell (or itsreceptor) engaged in the inflammatory or immune response, such as Tcells, macrophages or dendritic cells. Antibodies against an MHC classII invariant chain target, such as CD74, have been proposed by Bucala etal. (US 2003/0013122 A1), for treating MIF-regulated diseases, andHansen et al. (US 2004/0115193 A1) proposed at least one CD74 antibodyfor treating an immune dysregulation disease, an autoimmune disease,organ graft rejection, and graft-versus-host disease. Hansen et al.describe the use of fusion proteins of anti-CD74 with other antibodiesreacting with antigens/receptors on host cells such as lymphocytes andmacrophages for the treatment of such diseases. However, combinationswith targets other than CD74 are not suggested, and the disclosurefocuses on a different method of immunotherapy. Similar targets are alsouseful to treat atherosclerotic plaques (Burger-Kentischer et al.,Circulation 2002; 105:1561-1566).

In the treatment of infectious, autoimmune, organ transplantation,inflammatory, and graft-versus-host (and other immunoregulatory)diseases, diverse and relatively non-specific cytotoxic agents are usedto either kill or eliminate the noxient or microorganism, or to depressthe host's immune response to a foreign graft or immunogen, or thehost's production of antibodies against “self,” etc. However, theseusually affect the lymphoid and other parts of the hematopoietic system,giving rise to toxic effects to the bone marrow (hematopoietic) andother normal host cells. Particularly in sepsis, where animmunosuppressed status is encountered, use of immunosuppressivetherapies would be counter-indicated, so it is a goal to effect acareful balance between targeting and inhibiting key cells of theadaptive immune system while not depleting those involved with the hostmaintaining an active immune system.

A need exists for improved, more selective therapy of cancer and diverseimmune diseases, including sepsis and septic shock, inflammation,atherosclerosis, cachexia, graft-versus-host, and other immunedysregulatory disorders.

SUMMARY

Various embodiments concern well-tolerated methods which usecompositions comprising multispecific antibodies or a combination ofseparate antibodies in the therapy of various inflammatory andimmune-dysregulatory diseases, infectious diseases, pathologicangiogenesis and cancer. The multispecific antibodies or combinations ofantibodies are more effective than agents which react specifically withonly one target associated with these conditions. The antibodies reactwith one or more targets selected from the group consisting of (A)histones, (B) proinflammatory effectors of the innate immune system, (C)coagulation factors, (D) complement factors and complement regulatoryproteins, and (E) targets specifically associated with an inflammatoryor immune-dysregulatory disorder or with a pathologic angiogenesis orcancer, wherein the latter target is not (A), (B), (C) or (D). At leastone of the targets is (A), (B), (C) or (D). Targets of the adaptiveimmune system, such as specific dendritic cells, macrophages, NK cells,T cells, B cells and their specialized populations also may be selected.When the composition comprises a single multispecific antibody, thenCD74 may excluded as a target, unless combined with an anti-histoneantibody. Furthermore, when the composition comprises a combination ofseparate antibodies, combinations are excluded where one of theantibodies targets a B-cell antigen and the other antibody targets aT-cell, plasma cell, macrophage or inflammatory cytokine, unless used incombination with an anti-histone antibody. Combinations of separateantibodies are also excluded where one of the antibodies targets CD20and the other antibody targets C3b or CD40 or CD40L, except wherecombined with an anti-histone antibody of this invention.

When the composition comprises a combination of separate antibodies,combinations are excluded where one of the antibodies targets CD19,CD20, CD21, CD22, CD23 or CD80 and the other antibody targets acomplement factor. More particularly, combinations are excluded whereone of the antibodies targets CD19, CD20, CD21, CD22, CD23 or CD80 andthe other antibody targets C3b or CD40. However, any of these can becombined with an anti-histone antibody of this invention.

Targets for Therapy of Immune Dysregulatory Disease, Infectious Diseaseand Inflammatory Disease

The proinflammatory effector of the innate immune system may be aproinflammatory effector cytokine, a proinflammatory effector chemokineor a proinflammatory effector receptor. Suitable proinflammatoryeffector cytokine include MIF, HMGB-1 (high mobility group box protein1), TNF-α, IL-1, IL-4 (interleukin-4), IL-5 (interleukin-5), IL-6, IL-8,IL-12 (interleukin-12), IL-15 (interleukin-15), IL-17 (interleukin-17),IL-18 (interleukin-18), and IL-23 (interleukin-23). Examples ofproinflammatory effector chemokines include CCL19, CCL21, IL-8, MCP-1,RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, GRO-β, and Eotaxin.Proinflammatory effector receptors include IL-4R (interleukin-4receptor), IL-6R (interleukin-6 receptor), IL-13R (interleukin-13receptor), IL-15R (interleukin-15 receptor), IL-17R (interleukin-17receptor) and IL-18R (interleukin-18 receptor).

The multispecific antibody or combination of antibodies also may reactspecifically with at least one coagulation factor, particularly tissuefactor (TF), thrombomodulin, or thrombin. In other embodiments, themultispecific antibody or combination of antibodies reacts specificallywith at least one complement factor or complement regulatory protein. Inpreferred embodiments, the complement factor is selected from the groupconsisting of C3, C5, C3a, C3b, and C5a. In these embodiments, targetcombinations preferably do not include those in which the other antibodytargets CD19, CD20, CD21, CD22, CD23 or CD80 when the compositioncomprises a combination of separate antibodies. When the antibody reactsspecifically with a complement regulatory protein, the complementregulatory protein preferably is selected from the group consisting ofCD46, CD55, CD59 and mCRP.

In one embodiment, the composition comprises two or more antibodieswhich differ in specificity, each of which reacts specifically with adifferent proinflammatory effector of the innate immune system.Alternatively, the composition comprises two or more antibodies thatdiffer in specificity, each of which reacts specifically with adifferent coagulation factor. In another embodiment, the compositioncomprises two or more antibodies that differ in specificity, each ofwhich reacts specifically with a different complement factor orcomplement regulatory protein. In yet other embodiments, the two or moreantibodies react specifically with at least one proinflammatory effectorof the innate immune system and with at least one coagulation factor, orwith at least one proinflammatory effector of the innate immune systemand with at least one complement factor or complement regulatoryprotein, or with at least one complement factor or complement regulatoryprotein and with at least one coagulation factor, respectively.Alternatively, the multispecific antibody may react specifically withmore than one proinflammatory effector of the innate immune system, orwith more than one coagulation factor, or with more than one complementfactor or complement regulatory protein. Preferred are combinations ofthe above that include an anti-histone antibody of the currentinvention.

The two or more antibodies may react specifically with more than oneepitope of the same proinflammatory effector of the innate immune systemor more than one epitope of the same coagulation factor or more than oneepitope of the same complement factor or complement regulatory proteinor more than one epitope of a histone. In any of these embodiments, themultispecific antibody additionally may react with a target specificallyassociated with an inflammatory or immune-dysregulatory disorder or witha pathologic angiogenesis or cancer, which target is not an (A), (B),(C) or (D) target as defined above. In other embodiments, themultispecific antibody reacts with a target specifically associated withan inflammatory or immune-dysregulatory disorder or with a pathologicangiogenesis or cancer, and with one or more (A), (B), (C) or (D)targets as defined above. An example of a useful target for pathologicangiogenesis is Flt-1.

The composition alternatively may comprise at least one solublereceptor, or at least an extracellular domain of at least oneproinflammatory effector receptor. In one embodiment, the compositioncomprises at least one soluble receptor or at least an extracellulardomain of a proinflammatory effector receptor fused to at least oneantibody.

The composition may comprise at least one molecule reactive with aproinflammatory effector receptor. This molecule preferably is a naturalantagonist for the proinflammatory effector receptor, or a fragment ormutant of the antagonist that interacts specifically with the receptor.In one embodiment, the natural antagonist is the natural IL-1 receptorantagonist, or a fragment or mutant of this antagonist.

The multispecific antibody additionally may target dendritic cells,granulocytes, monocytes, macrophages, NK-cells, platelets, orendothelial cells. In some embodiments, the multispecific antibodyspecifically reacts with at least one antigen or receptor of theadaptive immune system. In other embodiments, the multispecific antibodyspecifically reacts with a cancer cell receptor, a cancer oncogene, orcancer-associated antigen, such as B-cell lineage antigens (CD19, CD20,CD21, CD22, CD23, etc.), VEGFR, EGFR, carcinoembryonic antigen (CEA),placental growth factor (PLGF), tenascin, HER-2/neu, EGP-1, EGP-2, CD25,CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD80, CD138, NCA66, MUC1,MUC2, MUC3, MUC4, MUC5ac, MUC16, IL-6, α-fetoprotein (AFP), A33, CA125,colon-specific antigen-p (CSAp), folate receptor, HLA-DR, humanchorionic gonadotropin (HCG), Ia, EL-2, insulin-like growth factor(ILGF) and ILGF receptor, KS-1, Le(y), MAGE, necrosis antigens, PAM-4mucin, MUC5ac, prostatic acid phosphatase (PAP), Pr1, prostate specificantigen (PSA), PSMA, S100, T101, TAC, TAG72, TRAIL receptors, orcarbonic anhydrase IX. Flt-3, which targets proliferating myeloid bonemarrow cells, also is a useful in identifying and treating certaincancers. Alternatively, the multispecific antibody may reactspecifically with a target such as C5a, Factor H, FHL-1, LPS, IFNγ orB7, or with a target such as CD2, CD4, CD14, CD18, CD11a, CD19, CD20,CD22, CD23, CD25, CD29, CD38, CD40L, CD52, CD64, CD83, CD147 or CD154.

When a proinflammatory effector receptor is targeted, in a preferredembodiment the actual target may be an extracellular domain of theproinflammatory effector receptor. This extracellular domain of theproinflammatory effector receptor may be fused to an antibody. Moreparticularly, the proinflammatory effector may be a soluble receptor orreceptor ligand which is fused to an antibody. In an alternativeembodiment, the composition may comprise at least one molecule reactivewith a proinflammatory effector receptor. This molecule may be a naturalantagonist for said proinflammatory effector receptor, or a fragment ormutant of this antagonist that interacts specifically with the receptor.In a preferred embodiment, the natural antagonist is the natural IL-1receptor antagonist, or a fragment or mutant of this antagonist.

One of the at least two different targets to which the multispecificantibody binds specifically may be a target that is not aproinflammatory effector of the immune system or a coagulation factor.In this case the multispecific antibody also binds specifically with atleast one proinflammatory effector of the immune system or at least onecoagulation factor. In one embodiment, this at least one other target isan antigen or receptor of the adaptive immune system. In otherembodiments, the at least one other target of the multispecific antibodytargets cells of the innate immune system, such as granulocytes,monocytes, macrophages, dendritic cells, and NK-cells. Other targetsinclude platelets and endothelial cells. Yet another group of targets isthe group consisting of C5a, LPS, IFN-γ and B7. A further group ofsuitable targets include CD2, CD4, CD14, CD18, CD11a, CD20, CD22, CD23,CD25, CD29, CD38, CD40L, CD52, CD64, CD83, CD147, and CD154. The CDs aretargets on immune cells, which can be blocked by antibodies to preventan immune cell response. CD83 is particularly useful as a marker ofactivated dendritic cells (Cao et al., Biochem J., Aug. 23, 2004 (Epubahead of print); Zinser et al., J. Exp Med. 200(3):345-51 (2004)).

Certain targets are of particular interest, such as MIF, HMGB-1, TNF-α,the complement factors and complement regulatory proteins, and thecoagulation factors. MIF is a pivotal cytokine in of the innate immunesystem and plays an important part in the control of inflammatoryresponses. Originally described as a T lymphocyte-derived factor thatinhibited the random migration of macrophages, the protein known asmacrophage migration inhibitory factor (MIF) was an enigmatic cytokinefor almost 3 decades. In recent years, the discovery of MIF as a productof the anterior pituitary gland and the cloning and expression ofbioactive, recombinant MIF protein have led to the definition of itscritical biological role in vivo. MIF has the unique property of beingreleased from macrophages and T lymphocytes that have been stimulated byglucocorticoids. Once released, MIF overcomes the inhibitory effects ofglucocorticoids on TNF-α, IL-1β, IL-6, and IL-8 production byLPS-stimulated monocytes in vitro and suppresses the protective effectsof steroids against lethal endotoxemia in vivo. MIF also antagonizesglucocorticoid inhibition of T-cell proliferation in vitro by restoringIL-2 and IFN-gamma production. MIF is the first mediator to beidentified that can counter-regulate the inhibitory effects ofglucocorticoids and thus plays a critical role in the host control ofinflammation and immunity. MIF is particularly useful in treatingcancer, pathological angiogenesis, and sepsis or septic shock, andtherefore a useful target to be combined with anti-histone antibodies ofthis invention.

HMGB-1, a DNA binding nuclear and cytosolic protein, is aproinflammatory cytokine released by monocytes and macrophages that havebeen activated by IL-1β, TNF, or LPS. Via its B box domain, it inducesphenotypic maturation of DCs. It also causes increased secretion of theproinflammatory cytokines IL-1α, IL-6, IL-8, IL-12, TNF-α and RANTES.HMGB-1 released by necrotic cells may be a signal of tissue or cellularinjury that, when sensed by DCs, induces and or enhances an immunereaction. Palumbo et al. report that HMGB-1 induces mesoangioblastmigration and proliferation (J Cell Biol, 164:441-449 (2004)).

HMGB-1 is a late mediator of endotoxin-induced lethality that exhibitssignificantly delayed kinetics relate to TNF and IL-1beta. Experimentaltherapeutics that target specific early inflammatory mediators such asTNF and IL-1 beta alone have not proven efficacious in the clinic, butmultispecific antibodies according to the present invention can improveresponse by targeting both early and late inflammatory mediators,especially when combined with the anti-histone antibodies of thisinvention.

Multispecific antibodies that target HMBG-1 are especially useful intreating arthritis, particularly collagen-induced arthritis.Multispecific antibodies comprising HMGB-1 also are useful in treatingsepsis and/or septic shock. Yang et al., PNAS USA 101:296-301 (2004);Kokkola et al., Arthritis Rheum, 48:2052-8 (2003); Czura et al., JInfect Dis, 187 Suppl 2:S391-6 (2003); Treutiger et al., J Intern Med,254:375-85 (2003).

TNF-α is an important cytokine involved in systemic inflammation and theacute phase response. TNF-α is released by stimulated monocytes,fibroblasts, and endothelial cells. Macrophages, T-cells andB-lymphocytes, granulocytes, smooth muscle cells, eosinophils,chondrocytes, osteoblasts, mast cells, glial cells, and keratinocytesalso produce TNF-α after stimulation. Its release is stimulated byseveral other mediators, such as interleukin-1 and bacterial endotoxin,in the course of damage, e.g., by infection. It has a number of actionson various organ systems, generally together with interleukins-1 and -6.One of the actions of TNF-α is appetite suppression; hence multispecificantibodies for treating cachexia preferably target TNF-α. It alsostimulates the acute phase response of the liver, leading to an increasein C-reactive protein and a number of other mediators. It also is auseful target when treating sepsis or septic shock, which is the basisfor its being combined with anti-histone antibodies and/orthrombomodulin in such diseases, particularly sepsis and autoimmunediseases.

The complement system is a complex cascade involving proteolyticcleavage of serum glycoproteins often activated by cell receptors. The“complement cascade” is constitutive and non-specific but it must beactivated in order to function. Complement activation results in aunidirectional sequence of enzymatic and biochemical reactions. In thiscascade, a specific complement protein, C5, forms two highly active,inflammatory byproducts, C5a and C5b, which jointly activate white bloodcells. This in turn evokes a number of other inflammatory byproducts,including injurious cytokines, inflammatory enzymes, and cell adhesionmolecules. Together, these byproducts can lead to the destruction oftissue seen in many inflammatory diseases. This cascade ultimatelyresults in induction of the inflammatory response, phagocyte chemotaxisand opsonization, and cell lysis.

The complement system can be activated via two distinct pathways, theclassical pathway and the alternate pathway. Most of the complementcomponents are numbered (e.g., C1, C2, C3, etc.) but some are referredto as “Factors.” Some of the components must be enzymatically cleaved toactivate their function; others simply combine to form complexes thatare active. Active components of the classical pathway include C1q, C1r,C1s, C2a, C2b, C3a, C3b, C4a, and C4b. Active components of thealternate pathway include C3a, C3b, Factor B, Factor Ba, Factor Bb,Factor D, and Properdin. The last stage of each pathway is the same, andinvolves component assembly into a membrane attack complex. Activecomponents of the membrane attack complex include C5a, C5b, C6, C7, C8,and C9n. Anti-C5a combined with anti-histone antibodies of thisinvention is particularly effective for the therapy of coagulopathiesand sepsis.

While any of these components of the complement system can be targetedby a multispecific antibody, certain of the complement components arepreferred. C3a, C4a and C5a cause mast cells to release chemotacticfactors such as histamine and serotonin, which attract phagocytes,antibodies and complement, etc. These form one group of preferredtargets according to the invention. Another group of preferred targetsincludes C3b, C4b and C5b, which enhance phagocytosis of foreign cells.Another preferred group of targets are the predecessor components forthese two groups, i.e., C3, C4 and C5. C5b, C6, C7, C8 and C9 inducelysis of foreign cells (membrane attack complex) and form yet anotherpreferred group of targets.

Complement C5a, like C3a, is an anaphylatoxin. It mediates inflammationand is a chemotactic attractant for induction of neutrophilic release ofantimicrobial proteases and oxygen radicals. Therefore, C5a and itspredecessor C5 are particularly preferred targets. By targeting C5, notonly is C5a affected, but also C5b, which initiates assembly of themembrane-attack complex. Thus, C5 is another preferred target. C3b, andits predecessor C3, also are preferred targets, as both the classicaland alternate complement pathways depend upon C3b. Three proteins affectthe levels of this factor, C1 inhibitor, protein H and Factor I, andthese are also preferred targets according to the invention. Complementregulatory proteins, such as CD46, CD55, and CD59, may be targets towhich the multispecific antibodies bind.

Coagulation factors also are preferred targets according to theinvention, particularly tissue factor (TF), thrombomodulin, andthrombin. TF is also known also as tissue thromboplastin, CD142,coagulation factor III, or factor III. TF is an integral membranereceptor glycoprotein and a member of the cytokine receptor superfamily.The ligand binding extracellular domain of TF consists of two structuralmodules with features that are consistent with the classification of TFas a member of type-2 cytokine receptors. TF is involved in the bloodcoagulation protease cascade and initiates both the extrinsic andintrinsic blood coagulation cascades by forming high affinity complexesbetween the extracellular domain of TF and the circulating bloodcoagulation factors, serine proteases factor VII or factor VIIa. Theseenzymatically active complexes then activate factor IX and factor X,leading to thrombin generation and clot formation.

TF is expressed by various cell types, including monocytes, macrophagesand vascular endothelial cells, and is induced by IL-1, TNF-α orbacterial lipopolysaccharides. Protein kinase C is involved in cytokineactivation of endothelial cell TF expression. Induction of TF byendotoxin and cytokines is an important mechanism for initiation ofdisseminated intravascular coagulation seen in patients withGram-negative sepsis. TF also appears to be involved in a variety ofnon-hemostatic functions including inflammation, cancer, brain function,immune response, and tumor-associated angiogenesis. Thus, multispecificantibodies that target TF are useful not only in the treatment ofcoagulopathies, but also in the treatment of sepsis, cancer, pathologicangiogenesis, and other immune and inflammatory dysregulatory diseasesaccording to the invention. A complex interaction between thecoagulation pathway and the cytokine network is suggested by the abilityof several cytokines to influence TF expression in a variety of cellsand by the effects of ligand binding to the receptor. Ligand binding(factor VIIa) has been reported to give an intracellular calcium signal,thus indicating that TF is a true receptor.

Thrombin is the activated form of coagulation factor II (prothrombin);it converts fibrinogen to fibrin. Thrombin is a potent chemotaxin formacrophages, and can alter their production of cytokines and arachidonicacid metabolites. It is of particular importance in the coagulopathiesthat accompany sepsis. Numerous studies have documented the activationof the coagulation system either in septic patients or following LPSadministration in animal models. Despite more than thirty years ofresearch, the mechanisms of LPS-induced liver toxicity remain poorlyunderstood. It is now clear that they involve a complex and sequentialseries of interactions between cellular and humoral mediators. In thesame period of time, gram-negative systemic sepsis and its sequallaehave become a major health concern, attempts to use monoclonalantibodies directed against LPS or various inflammatory mediators haveyielded only therapeutic failures, as noted elsewhere herein.Multispecific antibodies according to the invention that target boththrombin and at least one other target address the clinical failures insepsis treatment.

A recombinant form of thrombomodulin has been approved for treatment ofdisseminated intravascular coagulation (DIC) and is in phase II clinicaltrials for DIC associated with sepsis (Okamoto et al., 2012, Crit CareRes Pract, Epub 2012 Feb. 28). Thrombomodulin has a pivotal role in theprotein C system that is important in the pathogensis of sepsis (Leviand Van der Poll, Minerva Anestesiol Epub Dec. 17, 2012). Downregulationof thrombomodulin in sepsis causes impaired activation of protein C thatis central in the modulation of coagulation and inflammation (Levi andVan der Poll, Minerva Anestesiol Epub Dec. 17, 2012). Administration ofrecombinant thrombomodulin is reported to have a beneficial effect onrestoration of coagulation and improvement of organ failure (Levi andVan der Poll, Minerva Anestesiol Epub Dec. 17, 2012). A recentretrospective study confirmed that treatment with recombinantthrombomodulin was associated with reduced mortality in hospitalizedpatients with sepsis-induced DIC (Yamakawa et al., 2013, Intensive CareMed, Epub Jan. 30, 2013).

In other embodiments, the multispecific antibodies bind to a MHC classI, MHC class II or accessory molecule, such as CD40, CD54, CD80 or CD86.The multispecific antibody also may bind to a T-cell activationcytokine, or to a cytokine mediator, such as NF-κB.

Targets associated with sepsis and immune dysregulation and other immunedisorders include MIF, IL-1, IL-6, IL-8, CD74, CD83, and C5aR.Antibodies and inhibitors against C5aR have been found to improvesurvival in rodents with sepsis (Huber-Lang et al., FASEB J2002;16:1567-1574; Riedemann et al., J Clin Invest 2002; 110:101-108) andseptic shock and adult respiratory distress syndrome in monkeys (Hangenet al., J Surg Res 1989; 46:195-199; Stevens et al., J Clin Invest 1986;77:1812-1816). Thus, for sepsis, one of the at least two differenttargets preferably is a target that is associated with infection, suchas LPS/C5a. Other preferred targets include HMGB-1, TF, CD14, VEGF, andIL-6, each of which is associated with septicemia or septic shock.Preferred multispecific antibodies are those that target two or moretargets from HMGB-1, TF and MIF, such as MIF/TF, and HMGB-1/TF, as wellas HMGB-1 and histone, and MIF and histone.

In still other embodiments, one of the at least two different targetsmay be a target this is associated with graft versus host disease ortransplant rejection, such as MIF (Lo et al., Bone Marrow Transplant,30(6):375-80 (2002)). One of the at least two different targets also mayone that associated with acute respiratory distress syndrome, such asIL-8 (Bouros et al., PMC Pulm Med, 4(1):6 (2004), atherosclerosis orrestenosis, such as MIF (Chen et al., Arterioscler Thromb Vasc Biol,24(4):709-14 (2004), asthma, such as IL-18 (Hata et al., Int Immunol,Oct. 11, 2004 Epub ahead of print), a granulomatous disease, such asTNF-α (Ulbricht et al., Arthritis Rheum, 50(8):2717-8 (2004), aneuropathy, such as carbamylated EPO (erythropoietin) (Leist et al.,Science 305(5681):164-5 (2004), or cachexia, such as IL-6 and TNF-α.

Other targets include C5a, LPS, IFN-gamma, B7; CD2, CD4, CD14, CD18,CD11a, CD11b, CD11c, CD14, CD18, CD27, CD29, CD38, CD40L, CD52, CD64,CD83, CD147, CD154. Activation of mononuclear cells by certain microbialantigens, including LPS, can be inhibited to some extent by antibodiesto CD18, CD11b, or CD11c, which thus implicate β.sub.2-integrins(Cuzzola et al., J Immunol 2000; 164:5871-5876; Medvedev et al., JImmunol 1998; 160: 4535-4542). CD83 has been found to play a role ingiant cell arteritis (GCA), which is a systemic vasculitis that affectsmedium- and large-size arteries, predominately the extracranial branchesof the aortic arch and of the aorta itself, resulting in vascularstenosis and subsequent tissue ischemia, and the severe complications ofblindness, stroke and aortic arch syndrome (Weyand and Goronzy, N Engl JMed 2003; 349:160-169; Hunder and Valente, In: Inflammatory Diseases ofBlood Vessels. G. S. Hoffman and C. M. Weyand, eds, Marcel Dekker, NewYork, 2002; 255-265). Antibodies to CD83 were found to abrogatevasculitis in a SCID mouse model of human GCA (Ma-Krupa et al., J ExpMed 2004; 199:173-183), suggesting to these investigators that dendriticcells, which express CD83 when activated, are criticalantigen-processing cells in GCA. In these studies, they used a mouseanti-CD83 Mab (IgG1 clone HB15e from Research Diagnostics). CD154, amember of the TNF family, is expressed on the surface of CD4-positiveT-lymphocytes, and it has been reported that a humanized monoclonalantibody to CD 154 produced significant clinical benefit in patientswith active systemic lupus erythematosus (SLE) (Grammar et al., J ClinInvest 2003; 112:1506-1520). It also suggests that this antibody mightbe useful in other autoimmune diseases (Kelsoe, J Clin Invest 2003;112:1480-1482). Indeed, this antibody was also reported as effective inpatients with refractory immune thrombocytopenic purpura (Kuwana et al.,Blood 2004; 103:1229-1236).

In rheumatoid arthritis, a recombinant interleukin-1 receptorantagonist, IL-1Ra or anakinra (Kineret®), has shown activity (Cohen etal., Ann Rheum Dis 2004; 63:1062-8; Cohen, Rheum Dis Clin North Am 2004;30:365-80). An improvement in treatment of these patients, whichhitherto required concomitant treatment with methotrexate, is to combineanakinra with one or more of the anti-proinflammatory effector cytokinesor anti-proinflammatory effector chemokines (as listed above). Indeed,in a review of antibody therapy for rheumatoid arthritis, Taylor (CurrOpin Pharmacol 2003; 3:323-328) suggests that in addition to TNF, otherantibodies to such cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 andIL-18, are useful.

There are certain advantages when the multispecific antibody is anantibody that is at least bispecific, including rapid clearance from theblood. For example, the bispecific antibody may bind to a receptor or toits target molecule, such as for LPS, IL-1, IL-10, IL-6, MIF, HMGB1,TNF, IFN, tissue factor, thrombin, CD14, CD27, and CD134. Many of theseexist as both receptors and as soluble forms in the blood. Binding bythe bispecific antibodies results in rapid clearance from the blood, andthen targeting by the second arm of the fusion protein to another cell,such as a macrophage, for transport and degradation by the cell,especially the lysosomes. This is particularly effective when the secondtargeting arm is against an internalizing antigen, such as CD74,expressed by macrophages and dendritic cells. This is consistent withthe invention of Hansen, U.S. Pat. No. 6,458,933, but focusing herein oninflammatory cytokines and other immune modulation molecules andreceptors for immune-dysregulation diseases, and cancer antigens for theimmunotherapy of these cancers.

Preferred multispecific antibodies for the treatment of cancer includeantibodies to CD55 and to any of the cancer antigens identified above,antibodies to CD46 and to any of the above cancer antigens, antibodiesto CD59 and to any of the above cancer antigens, antibodies to MIF andto any of the above cancer antigens, antibodies to NF-κB and any of theabove cancer antigens, and antibodies to IL-6 and to any of the abovecancer antigens other than IL-6. These multispecific antibodies fortreating cancer may be antibody combinations or fusion proteins, giventogether or separately.

The multispecific antibody or antibody combination may be used inconjunction with one or more secondary therapeutics. This secondarytherapeutic may be one that affects a component of the innate immunesystem. Alternatively, it may affect a component of the adaptive immunesystem. The secondary therapeutic may also be a component that affectscoagulation, cancer, or an autoimmune disease, such as a cytotoxic drug.

The multispecific antibody may react specifically with targets ormarkers associated with specific diseases and conditions, such asinfectious diseases, acute respiratory distress syndrome, septicemia orseptic shock, graft versus host disease or transplant rejection,atherosclerosis, asthma, acne, giant cell arteritis, a granulomatousdisease, a neuropathy, cachexia, a coagulopathy such as diffuseintravascular coagulation (DIC), or myocardial ischemia.

The multispecific antibodies or antibody combinations are useful intreating conditions such as inflammatory or immune-dysregulatorydisorders, pathologic angiogenesis or cancer, and infectious disease.The composition can be used to treat septicemia or septic shock,infectious disease (bacterial, viral, fungal, or parasitic), neuropathy,graft versus host disease or transplant rejection, acute respiratorydistress syndrome, a granulomatous disease, asthma, atherosclerosis,acne, giant cell arteritis, coagulopathies such as diffuse intravascularcoagulation (DIC), or cachexia. In other embodiments, the condition isan autoimmune disease, especially a Class III autoimmune diseases.

The composition also can be used to treat a pathologic angiogenesis orcancer. The cancer may be hematopoietic cancer, such as leukemia,lymphoma, or myeloma, etc. Alternatively, the cancer may be a solidtumor, such as a carcinoma, melanoma, sarcoma, glioma, etc.

The subject antibody may be an immunoconjugate that comprises atherapeutic agent, such as a radionuclide, an immunomodulator, ahormone, a hormone antagonist, an enzyme, an enzyme inhibitor,oligonucleotide, a photoactive therapeutic agent, a cytotoxic agent, anantibody, an angiogenesis inhibitor, an immune modulator, and acombination thereof. When the therapeutic agent is an oligonucleotide itmay be an antisense oligonucleotide. Therapeutic agents are discussedabove in more detail.

The present invention also provides a method of treating a conditionselected from an inflammatory or immune-dysregulatory disorders, apathologic angiogenesis or cancer, and an infectious disease, comprisingadministering a therapeutically effective amount of a multispecificantibody that includes a hapten binding site, to a patient that issuspected of having such a condition; permitting the multispecificantibody to accrete at target sites; waiting for circulatingmultispecific antibody to clear from the bloodstream; administering tosaid subject a hapten that comprises a therapeutic agent; and allowingthe hapten with the therapeutic agent to bind to the hapten binding siteof said multispecific antibody. Preferably, the multispecific antibodyhas an anti-histone antibody as described in this invention as one ofthe included antibodies.

The multispecific antibodies described herein are useful for treatmentof autoimmune diseases, particularly for the treatment of Class IIIautoimmune diseases including immune-mediated thrombocytopenias, such asacute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myastheniagravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,polyglandular syndromes, bullous pemphigoid, diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.

The multispecific antibodies also are useful in treating inflammatory orimmune-dysregulatory disorders other than autoimmune disease. Examplesof these other inflammatory or immune-dysregulatory disorders that canbe treated with composition according to the invention includesepticemia or septic shock, infection, neuropathies, graft versus hostdisease, transplant rejection, acute respiratory distress syndrome,granulomatous disease, asthma, acne, diffuse intravascular coagulation(DIC), and atherosclerosis.

The multispecific antibodies also can be used in treating inflammationassociated with an infectious disease, including viral infections,bacterial infections, parasitic infections, and fungal infections.Exemplary viruses include the species of human immunodeficiency virus(HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus,hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningitis virus, wart virus and blue tongue virus. Exemplarybacteria include Anthrax bacillus, Streptococcus agalactiae, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzaeB, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis andClostridium tetani. Exemplary protozoans are Plasmodium falciparum,Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosomacruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosomamansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella,Onchocerca volvulus, Leishmania tropica, Trichinella spiralis,Onchocerca volvulus, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus or Mesocestoides corti.Exemplary mycoplasma are Mycoplasma arthritidis, Mycoplasma hyorhinis,Mycoplasma orale, Mycoplasma arginini, Acholeplasma laidlawii,Mycoplasma salivarum, and Mycoplasma pneumoniae. The fungus may be fromthe species of Microsporum, Trichophyton, Epidermophyton, Sporothrixschenckii, Cyrptococcus neoformans, Coccidioides immitis, Histoplasmacapsulatum, Blastomyces dermatitidis, or Candida albicans. Exemplaryparasites include malarial parasites, spirochetes and the like,including helminthes. Listings of representative disease-causinginfectious organisms to which antibodies can be developed for use inthis invention are contained in the second and subsequent editions ofDavis et al., MICROBIOLOGY (Harper & Row, New York, 1973 and later), andare well known to one of ordinary skill in the art. In theseembodiments, the multispecific antibody preferably targets an antigenassociated with the microbe or parasite.

Sepsis and septic shock are characterized by overwhelming inflammatoryand immune responses, which make them particularly susceptible totreatment with multispecific antibodies according to the presentinvention. Treatment of these conditions according to the presentinvention entails combining agents that work via different mechanisms,and preferably by administering fusion proteins of antagonist or agonistmediators or antibodies which function against more than one targetmolecule involved in the pathogenesis of this immune dysregulatory,inflammatory disease. As advocated by Van Amersfoort et al. (ibid.), “anattempt should be made to restore the balance between the pro- andanti-inflammatory responses.” The present invention restores the balanceand provides a clear improvement art over the use of single agents thatneutralize the proinflammatory cytokines, such TNF or IL-1, in patientswith sepsis, by using multispecific antibodies specific for at least twodifferent targets, where the targets are selected from the groupconsisting of proinflammatory effectors of the innate immune system,coagulation factors, and targets specifically associated with sepsis orseptic shock. More preferably, the multispecific construct contains ananti-histone antibody (or fragment), as provided in this invention.

In one embodiment for treatment of sepsis or septic shock, differentanti-inflammatory agents are combined with activated protein C, as wellas with anti-coagulation agents, such as thrombomodulin, and at leastone component of this multiple agent therapy is an agonist or antagonistantibody to at least one target receptor or mediator of inflammation orcoagulation, including complement pathway antagonists, and morepreferably an anti-histone antibody as provided in this invention. Alisting of selected anti-inflammatory and immunomodulating agents usedto treat patients with severe sepsis and septic shock is found in Bochudand Calandra (Brit Med J 2003; 326:262-266), and clinical trials of mostof these immunomodulatory therapies of severe sepsis and septic shockare reviewed in Vincent et al., Clin Infect Dis 2002; 34:1084-93.

Particularly preferred agents useful in treatment of sepsis and septicshock are multispecific antibodies that target MIF, LPS, TNF-α, C5a, C5areceptor (C5aR), TLR2 or HMGB-1 as one of the targets. The other targetcan also be selected from these, as well as from other proinflammatorycytokines or receptors, such as interleukin IL-1, TSST-1 (toxic shocksyndrome toxin 1), NCA-90, NCA-95, and HLA-DR. Preferred combinations ofagents or fusion proteins for treatment of severe sepsis or septic shockinclude those that target MIF and C5a receptor (C5aR), MIF and IL-6, LPSand MIF, TNF-α and HMGB-1, TLR2 (toll-like receptor-2) and LPS, TLR2 andIL-6, TLR2 and C5aR. An anti-MIF/anti-NCA-90 or an anti-MIF/anti-HLA-DRmultispecific antibody can be used to target granulocytes inblood/infectious deposits to neutralize MIF in patients with earlyevidence of toxic shock. These combinations can also includeanti-histone antibodies, as described herein, in various combinations,such as with antibodies against MIF, IL-6, C5a, TNF-alpha, LPS, orHMGB-1. Antibodies against CD74, such as milatuzumab, may also becombined with anti-histone antibodies, as described herein, for improvedtherapy of various inflammatory, immune dysregulatory, or malignant(cancerous) diseases. Still more preferable, for sepsis and septic shocktherapy (and the induced disseminated intravascular coagulation), is theaddition of thrombomodulin [rhTM] (such as recombinant human solublethrombomodulin (Yamakawa, Intensive Care Med 2013; published online 30Jan. 2013; DO1 10.1007/s00134-013-2822-2). Preferably the antibodystructures are humanized or human antibody constructs. These are readilycombined or constructed by those of skill in the art from availableantibodies. For example, T2.5 Mab has been developed as an antagonist toTLR-2 by immunizing a TLR2-neg mouse with TLR2 extracellular domain, andthis antibody inhibits release of inflammatory mediators, such as TNF-αand prevents lethal shock-like syndrome in mice (Meng et al., J ClinInvest 2004; 113:1473-1481). In preferred embodiments, recombinantactivated protein C is used as a secondary therapeutic in combinationwith antibody mixtures and fusion proteins. Likewise, as discussed,recombinant thrombomodulin is also used as a secondary therapeutic incombination with antibody mixtures and fusion proteins.

It also has been discovered that multispecific antibodies which targetboth a complement regulatory factor such as CD46, CD55, and/or CD59 anda tumor-associated antigen, and more particularly at least bispecificantibodies in which one arm targets the complement regulatory factor anda second arm targets an tumor associated antigen, are more effective intreating cancer than antibodies that target just one of these antigens.Moreover, contrary to the teaching of Sier et al., supra, it has beendiscovered that the use of beta-glucan is not obligatory in vivo for theimproved efficacy of a such a multispecific antibody over the use of theanticancer antibody alone, and that the bispecific antibody targetingthe cancer and the complement-regulatory protein (e.g., CD55) increasescancer cell killing over either antibody used by itself, specificallyagainst tumors that have a high expression of the complement-regulatoryprotein (thus blocking complement-mediated cytotoxicity by antibodies).

Another preferred complement-related target for neutralizing antibodiesis complement factor H (and its variant FHL-1) involved in thealternative pathway for complement, especially since factor H may beoverexpressed by some cancers (Ajona et al., Cancer Res 2004;64:6310-6318, and references cited therein). Therefore, use ofmultispecific antibodies, and particularly multispecific antibodies,directed against complement factor H and factor FHL-1 are of particularimportance. Multispecific antibodies against complement factor H and itsvariant FHL-1 additionally may target CD55, CD46 and/or CD59, as well asother complement factors. The targeting of these multispecificantibodies and to tumor-associated antigens and receptors has been foundto enhance specific targeting of complement antibodies to the tumorcells, and to provide an advantage over use of antibodies targeting asingle antigen or epitope. This has overcome the inconsistencies in theliterature published to date.

In non-malignant conditions, there is a different approach. Thisincludes neutralization or interference with other complement receptorsor factors, including complement-derived anaphylatoxin C5a orcomplement-receptor 3 (CR3, CD18/11b), which can mediate adhesion ofinflammatory cells to the vascular endothelium. In such situations,increased expression of CD46, CD55, and/or CD59 is desired in order tomitigate complement-mediated immunity, and also to reduce hyperacuterejection, as in organ transplant-rejection. Therefore, use of agonistsof such complement regulatory factors would be advantageous.

Particularly preferred agents useful in treatment of atherosclerosis aremultispecific antibodies that target MIF, low-density lipoprotein (LDL),and CEACAM6 (e.g., NCA-90). The other target can also be selected fromthese, as well as from other proinflammatory cytokines Preferredcombinations of agents or fusion proteins for treatment ofatherosclerosis target MIF and low-density lipoprotein-modifiedepitopes, NCA-90 and MIF, NCA-90 and low-density lipoprotein (LDL)epitopes, or LDL and CD83. There are readily combined or constructed bythose of skill in the art from commercially available antibodies. Forexample, Mab MDA2, a prototype Mab, recognizes malondialdehyde-lysingepitopes (e.g., in malondialdehyde-modified LDL) within oxidation-richatherosclerotic lesions (as described by Tsimikas et al., J Nucl Cardiol1999; 6:81-90).

In addition to sepsis and atherosclerosis, MIF has been reported to beexpressed in rabbits with atherogenesis (Lin et al., Circulation Res2000; 87:1202-1208), indicating that it is a key cytokine for thiscondition. Other diseases in which MIF has been implicated includeglomerulonephritis, arthritis, delayed-type hypersensitivity, gastricinflammation, and acute myocardial ischemia (reviewed by Yu et al., JHistochem Cytochem 2003; 625-631). Multispecific antibodies that targetMIF are therefore useful in treating any of these conditions.

As many as 500,000 individuals in the U.S. develop sepsis each year, anumber that is rising with the aging of the population. Despite the bestin antibiotic therapy and cardiopulmonary support, and the advances inunderstanding of inflammation and coagulation in sepsis, as many as halfthese cases are fatal. During infection, pro-inflammatory cytokines arereleased and activated. These include TNF-α, IL-1, and IL-6.Anti-inflammatory mediators, including IL-4 and IL-10, appearinsufficient to regulate pro-inflammatory cytokines in severe sepsis.

Prominent features of the septic response include uncontrolledinflammation and coagulation. Vascular endothelial damage is thetriggering event, whether caused by endotoxin, tissue factor, necroticcells, or amniotic fluid, becomes the triggering event. This endothelialdamage leads to release of tissue factor, which activates thecoagulation system resulting in excess thrombin generation. Subsequentclot formation promotes microvascular endothelial dysfunction, and, ifunchecked, hypoxemia, organ dysfunction, and organ failure ensue.

Endothelial damage and a shift towards a prothrombotic milieu lead todecreased expression and impaired function of endothelial receptors forthrombin, i.e., thrombomodulin, and protein C, i.e., the endothelialprotein C receptor (EPCR). Both thrombomodulin and EPCR are required forthe conversion of protein C to its active form, APC. Thus, a majorsystem for the regulation of thrombin formation, clot propagation, andprotein C activation is lost.

Nearly all patients with severe sepsis are deficient in protein C. Lowprotein C levels are associated with shock and poor outcomes, includingICU stay, ventilator dependence, and mortality. Supplying activatedprotein C exogenously in severe sepsis helps to restore regulation ofinflammatory and coagulation responses in some patients, leading to afavorable survival benefit. However, there is an obvious need for newtherapeutic modalities to reduce the procoagulant response, and preventseptic organ injury. A preferred secondary therapy is recombinant humanthrombomodulin (Yamakawa, 2013).

It has been established that blocking initiation of the procoagulantresponse before sepsis decreases mortality in nonhuman primates.Effective strategies to block initiation of extrinsic coagulation haveincluded use of monoclonal antibodies to TF, the natural TF pathwayinhibitor, and inactive analogs of FVIIa. In a recent study in baboons,it was demonstrated that blockade of the TF-VIIa complex with FVIIai atthe onset of sepsis attenuated sepsis-induced multiple organ injury anddramatically protected the lungs and kidneys. Antagonists that inhibitcomplement activation products, especially the anaphylatoxins, alsooffer promise to decrease sepsis mortality. C3a, C4a and C5a, appearduring sepsis, and the elevated anaphylotoxin plasma levels highlycorrelate with the development of multiorgan failure. In sepsis,complement may directly promote procoagulant activity or indirectlyinduce cytokine production. In vitro C5a and the terminal complex ofcomplement, C5b-9, induce tissue factor expression on endothelial cellsand monocytes, and assembly of C5b-9 on the surface of platelets hasbeen shown to stimulate prothrombinase activity. The present inventionprovides improved therapeutics for treating sepsis by providingmultispecific antibodies that target two or more of coagulation factors,proinflammatory cytokines and complement activations products.

The multispecific antibodies according to the invention bind to variousimmune or other host cells involved in the generation of inflammationand other immune-dysregulatory diseases (including intravascularcoagulation and myocardial ischemia). They also can be used to enhance ahost's immune response to cancer for cancer therapy or prevention. Inaddition, compositions and treatment methods are provided forneutralizing microbial toxins, such as LPS, neutralizingpro-inflammatory cytokines, and for overcoming abnormalities ofcoagulation. The methods use appropriate antibody combinations andfusion proteins directed against different participating factors in thecascade leading to severe sepsis, septic shock, and various otherimmune-dysregulatory diseases.

Although unconjugated multispecific antibodies and antibody fragmentsand mixtures of unconjugated antibodies and antibody fragments are thepreferred, primary therapeutic compositions for therapy according to theinvention, the efficacy of such therapy can be enhanced by supplementingthe multispecific antibodies with other therapies described herein. Insuch multimodal regimens, the supplemental therapeutic compositions canbe administered before, concurrently or after administration of themultispecific antibodies. For example, multimodal therapy of Class Mautoimmune diseases may comprise co-administration of therapeutics thatare targeted against T-cells, plasma cells or macrophages, such asantibodies directed against T-cell epitopes, more particularly againstthe CD4 and CD5 epitopes. Gamma globulins also may be co-administered.In some cases, it may be desirable to co-administer immunosuppressivedrugs such as corticosteroids and possibly also cytotoxic drugs. In thiscase, lower doses of the corticosteroids and cytotoxic drugs can be usedas compared to the doses used in conventional therapies, therebyreducing the negative side effects of these therapeutics. When thedisease to be treated is cancer, the use of various chemotherapeuticdrugs, naked antibodies used in immunotherapy, and radiation (externalor internal), can be combined with therapy according to the invention.Likewise, when infection and/or septicemia or septic shock are beingtreated, antimicrobial drugs may be used in combination with themultispecific antibodies.

Methods of Therapeutic Treatment

In various embodiments, antibodies or antigen-binding antibodyfragments, either alone or in combination with one or more othertherapeutic agents may be utilized. In certain embodiments, theantibodies or fragments thereof may be naked antibodies or fragments,that are not conjugated to any other therapeutic agents. In alternativeembodiments, the antibodies or fragments may be immunoconjugates thatare covalently attached to one or more therapeutic and/or diagnosticagents.

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats, comprising administering to the subject a therapeuticallyeffective amount of a cytotoxic immunoconjugate.

In one embodiment, immunological diseases which may be treated with thesubject anti-histone antibodies may include, for example, joint diseasessuch as ankylosing spondylitis, juvenile rheumatoid arthritis,rheumatoid arthritis; neurological disease such as multiple sclerosisand myasthenia gravis; pancreatic disease such as diabetes, especiallyjuvenile onset diabetes; gastrointestinal tract disease such as chronicactive hepatitis, celiac disease, ulcerative colitis, Crohn's disease,pernicious anemia; skin diseases such as psoriasis or scleroderma;allergic diseases such as asthma and in transplantation relatedconditions such as graft versus host disease and allograft rejection.

The administration of the cytotoxic immunoconjugates can be supplementedby administering concurrently or sequentially a therapeuticallyeffective amount of another antibody that binds to or is reactive withanother antigen on the surface of the target cell. Preferred additionalMAbs comprise at least one humanized, chimeric or human MAb selectedfrom the group consisting of a MAb reactive with CD4, CD5, CD8, CD14,CD15, CD16, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74,CD79a, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, B7,AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUC1,MUC2, MUC3, MUC4, MUC5AC, Ia, MIF, HM1.24, HLA-DR, tenascin, Flt-3,VEGFR, P1GF, ILGF, IL-6, IL-25, tenascin, TRAIL-R1, TRAIL-R2, complementfactor C5, oncogene product, or a combination thereof. Variousantibodies of use, such as anti-CD19, anti-CD20, and anti-CD22antibodies, are known to those of skill in the art. See, for example,Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., CancerImmunol. Immunother. 32:364 (1991); Longo, Curr. Opin. Oncol. 8:353(1996), U.S. Pat. Nos. 5,798,554; 6,187,287; 6,306,393; 6,676,924;7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785; 7,238,786;7,282,567; 7,300,655; 7,312,318; 7,501,498; 7,612,180; 7,670,804; andU.S. Patent Application Publ. Nos. 20080131363; 20070172920;20060193865; and 20080138333, the Examples section of each incorporatedherein by reference.

The anti-histone antibody therapy can be further supplemented with theadministration, either concurrently or sequentially, of at least onetherapeutic agent. For example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen usedto treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate, bendamustine, lenalidomide and bryostatin-1.

The subject anti-histone antibodies can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby theanti-histone antibody is combined in a mixture with a pharmaceuticallysuitable excipient. Sterile phosphate-buffered saline is one example ofa pharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The subject anti-histone antibodies can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Preferably, the anti-histone antibody is infused over a period of lessthan about 4 hours, and more preferably, over a period of less thanabout 3 hours. For example, the first 25-50 mg could be infused within30 minutes, preferably even 15 min, and the remainder infused over thenext 2-3 hrs. Formulations for injection can be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Additional pharmaceutical methods may be employed to control theduration of action of the anti-histone antibodies. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the anti-histone antibodies. For example, biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of releasefrom such a matrix depends upon the molecular weight of the anti-histoneantibody, the amount of anti-histone antibody within the matrix, and thesize of dispersed particles. Saltzman et al., Biophys. J. 55: 163(1989); Sherwood et al., supra. Other solid dosage forms are describedin Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The anti-histone antibody may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. Preferably, the anti-histone antibody is infused over a periodof less than about 4 hours, and more preferably, over a period of lessthan about 3 hours.

More generally, the dosage of an administered anti-histone antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. It may be desirable to provide the recipient with a dosage ofanti-histone antibody that is in the range of from about 0.1 mg/kg to 25mg/kg as a single intravenous infusion, although a lower or higherdosage also may be administered as circumstances dictate. A dosage of0.1-20 mg/kg for a 70 kg patient, for example, is 7-1,400 mg, or 4-824mg/m² for a 1.7-m patient. The dosage may be repeated as needed, forexample, once or twice per week for 4-10 weeks, once per week for 8weeks, or once per week for 4 weeks. It may also be given lessfrequently, such as every other week for several months, or monthly orquarterly for many months, as needed in a maintenance therapy.

Alternatively, an anti-histone antibody may be administered as onedosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.Or, the construct may be administered twice per week for 4-6 weeks. Ifthe dosage is lowered to approximately 200-300 mg/m² (340 mg per dosagefor a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may beadministered once or even twice weekly for 4 to 10 weeks. Alternatively,the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3months. It has been determined, however, that even higher doses, such as10 mg/kg once weekly or once every 2-3 weeks can be administered by slowi.v. infusion, for repeated dosing cycles. The dosing schedule canoptionally be repeated at other intervals and dosage may be giventhrough various parenteral routes, with appropriate adjustment of thedose and schedule.

In preferred embodiments, the anti-histone antibodies are of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor). Cancers conducive to treatment methods of thepresent invention involves cells which express, over-express, orabnormally express IGF-1R.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an antibody, antibody fragment, toxin or constituentfusion protein of an anti-histone antibody, such as a DNL™ construct.Fusion proteins may comprise an antibody or fragment or toxin attachedto, for example, an AD or DDD moiety.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine See Edelmanet al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. Pat.Nos. 7,531,327 and 7,537,930, the Examples section of each incorporatedherein by reference.

Autoimmune Disease

Exemplary autoimmune or immune dysfunction diseases include acute immunethrombocytopenia, chronic immune thrombocytopenia, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, pemphigus vulgaris, diabetes mellitus (e.g., juvenilediabetes), Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, fibrosing alveolitis, graft-versus-hostdisease (GVHD), organ transplant rejection, sepsis, septicemia andinflammation.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain one or more anti-histone antibodies as described herein. If thecomposition containing components for administration is not formulatedfor delivery via the alimentary canal, such as by oral delivery, adevice capable of delivering the kit components through some other routemay be included. One type of device, for applications such as parenteraldelivery, is a syringe that is used to inject the composition into thebody of a subject. Inhalation devices may also be used. In certainembodiments, a therapeutic agent may be provided in the form of aprefilled syringe or autoinjection pen containing a sterile, liquidformulation or lyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES Example 1 General Techniques for Construction of Anti-HistoneAntibodies

The Vκ (variable light chain) and V_(H) (variable heavy chain) sequencesfor anti-histone antibodies may be obtained by a variety of molecularcloning procedures, such as RT-PCR, 5′-RACE, and cDNA library screening.Specifically, the V genes of an anti-histone MAb from a cell thatexpresses a murine anti-histone MAb can be cloned by PCR amplificationand sequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized anti-histone MAb can then be designedand constructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine anti-histone MAb by general molecular cloningtechniques (Sambrook et al., Molecular Cloning, A laboratory manual,2^(nd) Ed (1989)). The Vκ sequence for the MAb may be amplified usingthe primers VH1BACK and VH1FOR (Orlandi et al., 1989) or the extendedprimer set described by Leung et al. (BioTechniques, 15: 286 (1993)).The V_(H) sequences can be amplified using the primer pairVH1BACK/VH1FOR (Orlandi et al., 1989) or the primers annealing to theconstant region of murine IgG described by Leung et al. (Hybridoma,13:469 (1994)).

PCR reaction mixtures containing 10 μl of the first strand cDNA product,10 μl of 10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mMMgCl₂, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.),250 μM of each dNTP, 200 nM of the primers, and 5 units of Taq DNApolymerase (Perkin Elmer Cetus) can be subjected to 30 cycles of PCR.Each PCR cycle preferably consists of denaturation at 94° C. for 1 min,annealing at 50° C. for 1.5 min, and polymerization at 72° C. for 1.5min. Amplified Vκ and VH fragments can be purified on 2% agarose(BioRad, Richmond, Calif.). The humanized V genes can be constructed bya combination of long oligonucleotide template syntheses and PCRamplification as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the Vκ PCR products. PCR products for V_(H) can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al. (Proc. Natl.Acad. Sci., USA, 74: 5463 (1977)).

Expression cassettes containing the Vκ and V_(H) sequences, togetherwith the promoter and signal peptide sequences, can be excised fromVKpBR and VHpBS, respectively, by double restriction digestion asHindIII-BamHI fragments. The Vκ and V_(H) expression cassettes can beligated into appropriate expression vectors, such as pKh and pG1g,respectively (Leung et al., Hybridoma, 13:469 (1994)). The expressionvectors can be co-transfected into an appropriate cell, e.g., myelomaSp2/0-Ag14 (ATCC, VA), colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of a chimeric, humanized orhuman anti-histone MAb by, for example, an ELISA assay. Alternatively,the Vκ and VH expression cassettes can be assembled in the modifiedstaging vectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHIfragments, respectively, and subcloned into a single expression vector,such as pdHL2, as described by Gilles et al. (J. Immunol. Methods125:191 (1989) and also shown in Losman et al., Cancer, 80:2660 (1997)).Another vector that is useful is the GS vector, as described in Barneset al., Cytotechnology 32:109-123 (2000). Other appropriate mammalianexpression systems are described in Werner et al., Arzneim.-Forsch./DrugRes. 48(11), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992). Following transfection, cells may be grown in 96-wellmicrotiter plates in complete HSFM medium (Life Technologies, Inc.,Grand Island, N.Y.) at 37° C., 5% CO₂. The selection process can beinitiated after two days by the addition of hygromycin selection medium(Calbiochem, San Diego, Calif.) at a final concentration of 500 units/mlof hygromycin. Colonies typically emerge 2-3 weeks post-electroporation.The cultures can then be expanded for further analysis. Transfectomaclones that are positive for the secretion of chimeric, humanized orhuman heavy chain can be identified by ELISA assay.

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M glycine buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA and its concentration adjusted to about 1 mg/mlusing PBS. Sodium azide, 0.01% (w/v), is conveniently added to thesample as preservative.

Example 2 Production of Chimeric IMMU-H4 (cIMMU-H4) Antibody

A chimeric form of the anti-H4 IMMU-H4 antibody was produced asdiscussed in Example 1 above. The variable region sequences used were asdisclosed in SEQ ID NO:98 and SEQ ID NO:99. The human heavy chainconstant region sequence used was as disclosed in SEQ ID NO:86. Thehuman light chain kappa constant region sequence was as disclosed inFIG. 7B of U.S. Pat. No. 7,151,164 (incorporated herein by reference).The purity of the cIMMU-H4 antibody was confirmed by SE-HPLCchromatography (not shown).

Representative data showing the binding affinity of cIMMU-H4 producedfrom a selected clone (4C3) are provided in FIG. 7. Surprisingly, thecIMMU-H4 (4C3) has a higher binding affinity (K_(D)=0.96 nM) forhistones than its murine counterpart (K_(D)=6.6 nM) based on ELISA.These surprising and unexpected results illustrate the superiority ofchimeric and humanized anti-histone antibodies compared to the parentalmurine antibodies.

Example 3 Production of Humanized IMMU-H4 Antibody

A humanized form of the anti-H4 IMMU-H4 antibody is produced accordingto Leung et al., (1995, Molec Immunol 32:1413-27). The variable regionsequences used are as disclosed in SEQ ID NO:98 an6 SEQ ID NO:97. Thehuman heavy chain constant region sequence used is as disclosed in SEQID NO:86. The human light chain kappa constant region sequence is asdisclosed in FIG. 7B of U.S. Pat. No. 7,151,164 (incorporated herein byreference).

The binding characteristics of the humanized IMMU-H4 antibody areidentical to those of the chimeric IMMU-H4 antibody.

Example 4 Production of Chimeric IMMU-H3 Antibody

A chimeric form of the anti-H3 IMMU-H3 antibody is produced as discussedin Example 1 above. The variable region sequences used are as disclosedin SEQ ID NO:108 and SEQ ID NO:109. The human heavy chain constantregion sequence used is as disclosed in SEQ ID NO:86. The human lightchain kappa constant region sequence is as disclosed in FIG. 7B of U.S.Pat. No. 7,151,164 (incorporated herein by reference). The chimericIMMU-H3 antibody competes for binding to H3 with, and binds to the sameepitope of H3 as, the parental murine antibody.

Example 5 Production of Humanized IMMU-H3 Antibody

A humanized form of the anti-H3 IMMU-H3 antibody is produced accordingto Leung et al., (1995, Molec Immunol 32:1413-27). The variable regionsequences used are as disclosed in SEQ ID NO:106 and SEQ ID NO:107. Thehuman heavy chain constant region sequence used is as disclosed in SEQID NO:86. The human light chain kappa constant region sequence is asdisclosed in FIG. 7B of U.S. Pat. No. 7,151,164 (incorporated herein byreference).

The binding characteristics of the humanized IMMU-H3 antibody areidentical to those of the chimeric IMMU-H3 antibody.

Example 6 Production of Chimeric IMMU-H2B Antibody

A chimeric form of the anti-H2B IMMU-H2B antibody is produced asdiscussed in Example 1 above. The variable region sequences used are asdisclosed in SEQ ID NO:118 and SEQ ID NO:119. The human heavy chainconstant region sequence used is as disclosed in SEQ ID NO:86. The humanlight chain kappa constant region sequence is as disclosed in FIG. 7B ofU.S. Pat. No. 7,151,164 (incorporated herein by reference). The chimericIMMU-H2B antibody competes for binding to H2B with, and binds to thesame epitope of H2B as, the parental murine antibody.

Example 7 Production of Humanized IMMU-H2B Antibody

A humanized form of the anti-H2B IMMU-H2B antibody is produced accordingto Leung et al., (1995, Molec Immunol 32:1413-27). The variable regionsequences used are as disclosed in SEQ ID NO:116 and SEQ ID NO:117. Thehuman heavy chain constant region sequence used is as disclosed in SEQID NO:86. The human light chain kappa constant region sequence is asdisclosed in FIG. 7B of U.S. Pat. No. 7,151,164 (incorporated herein byreference).

The binding characteristics of the humanized IMMU-H2B antibody areidentical to those of the chimeric IMMU-H2B antibody.

Example 8 Treatment of Septic Shock

M.N. is a 62-year-old white male with a history of chronic lymphocyticleukemia having past therapy with various cytotoxic drugs,corticosteroids, as well as rituximab and bendamustine, and presentingwith stable disease and a past history of several infections thatrequired prolonged antibiotic therapy. He is admitted to the emergencydepartment after being evaluated by his family physician as havingsymptoms of sepsis, with high temperature (40.7° C.), chills, dyspnea,palpitations, agitation, some confusion, nausea, and cool extremities.Examination reveals tachycardia (>100/min), hypotension (95/55 mm Hg),especially upon standing, and a reduced urine output (800 mL/d), andsigns of pneumonia. Tests show a low oxygen tension and acidosis, ablood count not detecting infection, but instead neutropenia (2,500WBC/mL, with 10% bands), platelets of 38,000, Hg of 6 g/dL, chest x-rayshowing a generalized pneumonia, blood tests indicate reduced renalfunction, with abnormal serum creatinine (3 mg/dL) and elevated BUNlevels, and elevated serum lactate indicating tissue hypoperfusion.Blood cultures reveal the presence of S. aureus and Gram-negativebacteria, supporting the diagnosis of septicemia. The patient also haslaboratory evidence of a coagulopathy presenting as sepsis-induceddisseminated intravascular coagulation (DIC), particularly affecting theextremities and his lungs. The patient is treated in the intensive careunit for severe sepsis and septic shock, which includes generalsupportive care (oxygen), hemodynamic support by fluid infusion torestore circulating blood volume (500 mL 0.9% sodium chloride andlactated Ringer solution, with up to 2.5 L given over first few hours),vasopressor supportive therapy with dopamine (Intropin, 3 mcg/kg/miniv), and antibiotic therapy with 400 mg IV every 12 hrs of ciprofloxacin(Cipro). The patient is also given recombinant human thrombomodulin(Recomodulin®) at 0.06 mg/kg per day, for a period of 6 days. Five daysafter admission, the patient is stable but does not show any significantimprovement in signs or symptoms, only slightly better urine excretion,a small rise in blood pressure, a small drop in temperature to 39.3° C.and an improvement of his International Society of Thrombosis andHemostasis (ISTH) DIC scores, including improvement in platelet count,prothrombin time, and fibrinogen level. The evidence of respiratorytract hemorrhage also appears to improve slightly, also with a reductionof his dyspnea. The patient is then given a combination of two humanizedmonoclonal antibodies sequentially twice weekly for 3 weeks, consistingof 300 mg humanized anti-MIF and 400 mg chimeric anti-histone (IMMU-H4),both by slow infusions over 4 hrs. On week 5 thereafter, the patientshows less confusion, a further drop in temperature, reduction oftachycardia, dyspnea, further improvement of DIC signs, and reducedpneumonia by both physical exam and chest x-ray. At the end of the 6thweek, his renal function tests also show some improvement (BUN and serumcreatinine values), and he is removed from the intensive care unit to aninfectious disease bed, with supportive care adjusted. Two months later,the patient receives a repeated cycle of thrombomodulin and thehumanized anti-MIF and chimeric anti-histone antibodies, as well as arepeated course of a broad-spectrum antibiotic, and then shows furtherimprovement so that he becomes ambulatory and has virtually normalmental function and an overall 70+% reduction of pneumonia and a feverof 38° C., and about an 85% normal urine output.

Example 9 Therapy of Systemic Lupus Erythematosus (SLE)

B. S. is a 35-year-old African-American female diagnosed 2 years earlierwith SLE, when she presented with a globerulonephritis (WHO grade 3),serositis, polyarthritis, and a vasculitic rash. She had prior therapywith corticosteroids (range of 15-60 mg prednisone per day) andhyrdoxychloroquine (200 mg/day), and at a later time also azathioprine(100 mg/day) and a course of methotrexate because of persistent disease.Over the years, she experiences flares of her SLE, presenting withpolyarthritis, lethargy, skin rash, and serositis. She now presents withpersistently active disease (BILAG A for musculoskeletal and BILAG B forcardiorespiratory systems, and BILAG C for other systems) andunresponsive to conventional therapies, but is maintained on 40 mgprednisone daily. She is given humanized anti-CD22 monoclonal antibody,epratuzumab, at 600 mg i.v. over 1 hr, repeated once in each of thefollowing four weeks. Four weeks after the third infusion, hercirculating B-lymphocytes are reduced by 40% from baseline prior totherapy, but her Hg level has risen from 8 g/dL to 10 g/dL. Her rash andpolyarthritis show some improvement, and her musculoskeletal systemBILAG A level is reduced to BILAG B, yet she requires additionaltherapy. At 8 weeks following her anti-CD22 antibody therapy, she isgiven a course of a bispecific antibody fusion protein consisting of arecombinant heteroconjugate of an anti-CD74 and an anti-histone(IMMU-H4) antibody, at a dose of 500 mg i.v. weekly times 4 weeks. Atevaluation at 2 months later, she has a marked improvement in all organsystems, to a BILAG C and D status in most, and is capable of having herprednisone dose tapered to 6.5 mg per day. At follow-up of 3 months,most of her organ symptoms remain stable, and she remains on this lowdoes of prednisone without any flare.

Example 10 Therapy of Non-Hodgkin's Lymphoma (NHL)

T M is a 68-year-old white male with a history of diffuse large B-cellNHL that has relapsed after therapy with standard cycles of CHOPchemotherapy and rituximab, and is now presenting with fever, lung andmediastinal infiltrates, enlarged cervical and axillary lymph nodes,spleen, and evidence of bone marrow involvement based on aspiration andcytology. He receives 6 weekly infusions of two humanized antibodies,one against histone (IMMU-H4) and the other against CD20 (veltuzumab),each given on the same day sequentially, over a 3-4-hr infusion foreach, at a dose of each of 200 mg. Four days after the last infusion,his examination indicates no major toxicities to the therapy, and somepalpable softening of his cervical and axillary lymph nodes, and areduction in the size of his spleen by palpation. At the next follow-upexamination in 8 weeks, almost all of his cervical and about half ofthese axillary nodes have disappeared, including normalization of thespleen size, and his chest x-ray and CT scan show evidence of about a50% shrinkage of his pulmonary and mediastinal infiltrates. About 4months later, his examination reveals that although his lymph node andpulmonary involvement are stable, there is a suggested increase in bonemarrow involvement and a drop in his Hg to 7 g/dL and a platelet fall to55,000/4. He then receives a bispecific antibody consisting of a fusedhumanized antibody against MIF and humanized antibody against histone(IMMU-H4 antibody), given twice weekly for 3 weeks at a dose of 200 mgper slow i.v. infusion. At his 3-month evaluation, his Hg shows a riseto 11 g/dL and his platelets rise to 120,000/4, there is a remarkabledecrease of NHL cells in the bone marrow aspirate, and there are nolymph nodes palpable or disease visible in the chest by radiologicalexaminations. The patient's response remains stable for another 4months.

Example 11 Therapy of Cancer-Related Cachexia

J.M. is a 68-year-old Caucasian male with a history of heavy cigarettesmoking and an inoperable small-cell lung cancer affecting his rightlung and paraortic and parabronchial lymph nodes on both sides. He hasreceived combination chemotherapy, which has shown myelotoxicities andevidence of some minor tumor shrinkage, being less than 30% of allmeasurable volume. He presents with considerable weight loss, beingalmost 2 meters high and now weighing 58 kg, suffering fromcancer-related cachexia. He is infused weekly for 8 weeks with ahumanized bispecific fusion antibody construct targeting both IL-6 andhistone (IMMU-H4 antibody), at a dose of 120 mg weekly. During the last2 weeks, his appetite improves, and he shows a weight gain to 65 kg at 7weeks post therapy, with more muscle strength and generally improvedvigor, which then remains stable at 70-75 kg over the next 2 months,when he begins to show progression of his malignant disease. Other thanhis cyclic chemotherapy, no corticosteroids were given during theantibody therapy, and he is considered to have responded to thistreatment for cachexia.

Example 12 Therapy of Immune Thrombocytopenia (ITP)

S.R. is a 32-year-old female who has a history of spontaneous for 2years and has been responsive to corticosteroid therapy with prednisonegiven at high doses for several courses. She now presents with severeITP, bruising and petecchiae, and a platelet count of 12,000/μL. Shealso complains of frequent gum and nose bleeding. She is given fourdoses of dexamethasone (40 mg) over two weeks, combined with a humanizedbispecific antibody construct made by DNL™ from humanized anti-CD20(veltuzumab) and humanized anti-histone (IMMU-H4) antibodies at a weeklysubcutaneous dose of 200 mg for three weeks. Blood count examination 2weeks later indicates a reduction of B cells by 80% and an increase inplatelets to 38,000/4, with reduced bruising, petecchiae, and bleedingmanifestations. Two weeks later, her platelets rise further to55,000/uL. She returns 2 months later for a repeated cycle of thistherapy, and then shows a rise of her platelets to 100,000/μL,considered as a complete response. This level is maintained for 3months, when tested last.

What is claimed is:
 1. A method of treating rapidly progressiveglomerulonephritis (RPG), comprising administering to a subject with RPGa chimeric or humanized anti-histone H4 antibody or antigen-bindingfragment thereof, comprising the heavy chain complementarity-determiningregion (CDR) sequences CDR1 (DDYLH, SEQ ID NO:90), CDR2(WIGWIDPENGDTEYASKFQG, SEQ ID NO:91) and CDR3 (PLVHLRTFAY, SEQ ID NO:92)and the light chain CDR sequences CDR1 (RASESVDSYDNSLH, SEQ ID NO:93),CDR2 (LASNLES, SEQ ID NO:94) and CDR3 (QQNNEDPWT, SEQ ID NO:95).
 2. Themethod of claim 1, wherein the antibody or fragment thereof is achimeric antibody or fragment and wherein the amino acid sequence of theheavy chain variable region sequence is SEQ ID NO:98 and the amino acidsequence of the light chain variable region sequence is SEQ ID NO:99. 3.The method of claim 1, wherein the antibody or fragment thereof is ahumanized antibody or fragment and wherein the amino acid sequence ofthe heavy chain variable region sequence is SEQ ID NO:96 and the aminoacid sequence of the light chain variable region sequence is SEQ IDNO:97.